Sulfamide linker, conjugates thereof, and methods of preparation

ABSTRACT

The present invention relates to a compound comprising an alpha-end and an omega-end, the compound comprising on the alpha-end a reactive group Q 1  capable of reacting with a functional group F 1  present on a biomolecule and on the omega-end a target molecule, the compound further comprising a group according to formula (1) or a salt thereof: 
     
       
         
         
             
             
         
       
     
     Said compound may also be referred to as a linker-conjugate. The invention also relates to a process for the preparation of a bioconjugate, the process comprising the step of reacting a reactive group Q 1  of a linker-conjugate according to the invention with a functional group F 1  of a biomolecule. The invention further relates to a bioconjugate obtainable by the process according to the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application filed under 35 U.S.C.§111(a) claiming the benefit under 35 U.S.C. §§120 and 365(c) ofInternational Application No. PCT/NL2015/050697 filed on Oct. 5, 2015,which is based upon and claims the benefit of priority of EuropeanPatent Application No. 14187615.1, filed on Oct. 3, 2014, the entirecontents of which are all hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention is in the field of bioconjugation. The inventionrelates to sulfamide linkers and conjugates thereof, and to methods forthe preparation thereof. More particularly, the invention relates tolinkers comprising an acylsulfamide group and/or a carbamoyl sulfamidegroup and to conjugates comprising said linkers. The invention furtherrelates to a process for the preparation of bioconjugates comprising alinker, the linker comprising an acylsulfamide group and/or a carbamoylsulfamide group.

BACKGROUND OF THE INVENTION

Bioconjugation is the process of linking two or more molecules, of whichat least one is a biomolecule. The biomolecule(s) may also be referredto as “biomolecule(s) of interest”, the other molecule(s) may also bereferred to as “target molecule” or “molecule of interest”. Typicallythe biomolecule of interest (BOI) will consist of a protein (orpeptide), a glycan, a nucleic acid (or oligonucleotide), a lipid, ahormone or a natural drug (or fragments or combinations thereof). Theother molecule of interest (MOI) may also be a biomolecule, henceleading to the formation of homo- or heterodimers (or higher oligomers),or the other molecule may possess specific features that are impartedonto the biomolecule of interest by the conjugation process. Forexample, the modulation of protein structure and function by covalentmodification with a chemical probe for detection and/or isolation hasevolved as a powerful tool in proteome-based research and biomedicalapplications. Fluorescent or affinity tagging of proteins is key tostudying the trafficking of proteins in their native habitat. Vaccinesbased on protein-carbohydrate conjugates have gained prominence in thefight against HIV, cancer, malaria and pathogenic bacteria, whereascarbohydrates immobilized on microarrays are instrumental in elucidationof the glycome. Synthetic DNA and RNA oligonucleotides (ONs) require theintroduction of a suitable functionality for diagnostic and therapeuticapplications, such as microarray technology, antisense andgene-silencing therapies, nanotechnology and various materials sciencesapplications. For example, attachment of a cell-penetrating ligand isthe most commonly applied strategy to tackle the low internalizationrate of ONs encountered during oligonucleotide-based therapeutics(antisense, siRNA). Similarly, the preparation of oligonucleotide-basedmicroarrays requires the selective immobilization of ONs on a suitablesolid surface, e.g. glass.

There are numerous examples of chemical reactions suitable to covalentlylink two (or more) molecular structures. However, labeling ofbiomolecules poses high restrictions on the reaction conditions that canbe applied (solvent, concentration, temperature), while the desire ofchemoselective labeling limits the choice of reactive groups. Forobvious reasons, biological systems generally flourish best in anaqueous environment meaning that reagents for bioconjugation should besuitable for application in aqueous systems. In general, two strategicconcepts can be recognized in the field of bioconjugation technology:(a) conjugation based on a functional group already present in thebiomolecule of interest, such as for example a thiol, an amine, analcohol or a hydroxyphenol unit or (b) a two-stage process involvingengineering of one (or more) unique reactive groups into a BOI prior tothe actual conjugation process.

The first approach typically involves a reactive amino acid side-chainin a protein (e.g. cysteine, lysine, serine and tyrosine), or afunctional group in a glycan (e.g. amine, aldehyde) or nucleic acid(e.g. purine or pyrimidine functionality or alcohol). As summarizedinter alia in G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rdEd. 2013, incorporated by reference, a large number of reactivefunctional groups have become available over the years forchemoselective targeting of one of these functional groups, such asmaleimide, haloacetamide, activated ester, activated carbonate, sulfonylhalide, activated thiol derivative, alkene, alkyne, allenamide and more,each of which requiring its own specific conditions for conjugation (pH,concentration, stoichiometry, light, etc.). Most prominently,cysteine-maleimide conjugation stands out for protein conjugation byvirtue of its high reaction rate and chemoselectivity. However, when nocysteine is available for conjugation, as in many proteins and certainlyin other biomolecules, other methods are often required, each sufferingfrom its own shortcomings.

An elegant and broadly applicable solution for bioconjugation involvesthe two-stage approach. Although more laborious, two-stage conjugationvia engineered functionality typically leads to higher selectivity(site-specificity) than conjugation on a natural functionality. Besidesthat, full stability can be achieved by proper choice of construct,which can be an important shortcoming of one stage conjugation on nativefunctionality, in particular for cysteine-maleimide conjugation. Typicalexamples of a functional group that may be imparted onto the BOI include(strained) alkyne, (strained) alkene, norbornene, tetrazine, azide,phosphine, nitrile oxide, nitrone, nitrile imine, diazo compound,carbonyl compound, (O-alkyl)hydroxylamine and hydrazine, which may beachieved by either chemical or molecular biology approach. Each of theabove functional groups is known to have at least one reaction partner,in many cases involving complete mutual reactivity. For example,cyclooctynes react selectively and exclusively with 1,3-dipoles,strained alkenes with tetrazines and phosphines with azides, leading tofully stable covalent bonds. However, some of the above functionalgroups have the disadvantage of being highly lipophilic, which maycompromise conjugation efficiency, in particular in combination with alipophilic molecule of interest (see below).

The final linking unit between the biomolecule and the other molecule ofinterest should preferentially also be fully compatible with an aqueousenvironment in terms of solubility, stability and biocompatibility. Forexample, a highly lipophilic linker may lead to aggregation (during orafter conjugation), which may significantly increase reaction timesand/or reduce conjugation yields, in particular when the MOI is also ofhydrophobic nature. Similarly, highly lipophilic linker-MOI combinationmay lead to unspecific binding to surfaces or specific hydrophobicpatches on the same or other biomolecules. If the linker is susceptibleto aqueous hydrolysis or other water-induced cleavage reactions, thecomponents comprising the original bioconjugate separate by diffusion.For example, certain ester moieties are not suitable due tosaponification while β-hydroxycarbonyl or γ-dicarbonyl compounds couldlead to retro-aldol or retro-Michael reaction, respectively. Finally,the linker should be inert to functionalities present in thebioconjugate or any other functionality that may be encountered duringapplication of the bioconjugate, which excludes, amongst others, the useof linkers featuring for example a ketone or aldehyde moiety (may leadto imine formation), an α,β-unsaturated carbonyl compound (Michaeladdition), thioesters or other activated esters (amide bond formation).

Compounds made of linear oligomers of ethylene glycol, so-called PEG(polyethyleneglycol) linkers, enjoy particular popularity nowadays inbiomolecular conjugation processes. PEG linkers are highly watersoluble, non-toxic, non-antigenic, and lead to negligble or noaggregation. For this reason, a large variety of linear, bifunctionalPEG linkers are commercially available from various sources, which canbe selectively modified at either end with a (bio)molecule of interest.PEG linkers are the product of a polymerization process of ethyleneoxide and are therefore typically obtained as stochastic mixtures ofchain length, which can be partly resolved into PEG constructs with anaverage weight distribution centered around 1, 2, 4 kDa or more (up to60 kDa). Homogeneous, discrete PEGs (dPEGs) are also known withmolecular weights up to 4 kDa and branched versions thereof go up to 15kDa. Interestingly, the PEG unit itself imparts particularcharacteristics onto a biomolecule. In particular, protein PEGylationmay lead to prolonged residence in vivo, decreased degradation bymetabolic enzymes and a reduction or elimination of proteinimmunogenicity. Several PEGylated proteins have been FDA-approved andare currently on the market.

By virtue of its high polarity, PEG linkers are perfectly suitable forbioconjugation of small and/or water-soluble moieties under aqueousconditions. However, in case of conjugation of hydrophobic, nonwater-soluble molecules of interest, the polarity of a PEG unit may beinsufficient to offset hydrophobicity, leading to significantly reducedreaction rates, lower yields and induced aggregation issues. In suchcase, lengthy PEG linkers and/or significant amounts of organiccosolvents may be required to solubilize the reagents. For example, inthe field of antibody-drug conjugates, the controlled attachment of adistinct number of toxic payloads to a monoclonal antibody is key, witha payload typically selected from the group of auristatins E or F,maytansinoids, duocarmycins, calicheamicins or pyrrolobenzodiazepines(PBDs), with many others are underway. With the exception of auristatinF, all toxic payloads are poorly to non water-soluble, whichnecessitates organic cosolvents to achieve successful conjugation, suchas 25% dimethylacetamide (DMA) or 50% propylene glycol (PG). In case ofhydrophobic payloads, despite the use of aforementioned cosolvents,large stoichiometries of reagents may be required during conjugationwhile efficiency and yield may be significantly compromised due toaggregation (in process or after product isolation), as for exampledescribed by Senter et al. in Nat. Biotechn. 2014, 24, 1256-1263,incorporated by reference. The use of long PEG spacers (12 units ormore) may partially enhance solubility and/or conjugation efficiency,but it has been shown that long PEG spacers may lead to more rapid invivo clearance, and hence negatively influence the pharmacokineticprofile of the ADC.

From the above, it becomes clear that there is a demand for short, polarspacers that enable the fast and efficient conjugation of hydrophobicmoieties. It is clear that the latter pertains even to a higher level onconjugation reactions where a hydrophobic reactive moiety is employed,such as for example strained alkynes, alkenes, and phosphines (seeabove).

Linkers are known in the art, and disclosed in e.g. WO 2008/070291,incorporated by reference. WO 2008/070291 discloses a linker for thecoupling of targeting agents to anchoring components. The linkercontains hydrophilic regions represented by polyethylene glycol (PEG)and an extension lacking chiral centers that is coupled to a targetingagent.

WO 01/88535, incorporated by reference, discloses a linker system forsurfaces for bioconjugation, in particular a linker system having anovel hydrophilic spacer group. The hydrophilic atoms or groups for usein the linker system are selected from the group consisting of O, NH,C═O (keto group), O—C═O (ester group) and CR³R⁴, wherein R³ and R⁴ areindependently selected from the group consisting of H, OH, C₁-C₄ alkoxyand acyloxy.

WO 2014/100762, incorporated by reference, describes compounds with ahydrophilic self-immolative linker, which is cleavable under appropriateconditions and incorporates a hydrophilic group to provide bettersolubility of the compound. The compounds comprise a drug moiety, atargeting moiety capable of targeting a selected cell population, and alinker which contains an acyl unit, an optional spacer unit forproviding distance between the drug moiety and the targeting moiety, apeptide linker which can be cleavable under appropriate conditions, ahydrophilic self-immolative linker, and an optional secondself-immolative spacer or cyclization self-elimination linker. Thehydrophilic self-immolative linker is e.g. a benzyloxycarbonyl group.

SUMMARY OF THE INVENTION

The invention relates to a compound (also referred to as alinker-conjugate) comprising an alpha-end and an omega-end, the compoundcomprising on the alpha-end a reactive group Q¹ capable of reacting witha functional group F¹ present on a biomolecule and on the omega-end atarget molecule, the compound further comprising a group according toformula (1) or a salt thereof:

wherein:

-   -   a is 0 or 1; and    -   R¹ is selected from the group consisting of hydrogen, C₁-C₂₄        alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl        groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups, the C₁-C₂₄ alkyl groups, C₃-C₂₄        cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups, or R¹ is a target molecule D, wherein the        target molecule is optionally connected to N via a spacer        moiety;        and wherein the group according to formula (1), or the salt        thereof, is situated in between said alpha-end and said        omega-end of the compound.

More in particular, the invention relates to a linker-conjugateaccording to formula (4a) or (4b), or a salt thereof:

wherein:a is independently 0 or 1;b is independently 0 or 1;c is 0 or 1;d is 0 or 1;e is 0 or 1;f is an integer in the range of 1 to 150;g is 0 or 1;i is 0 or 1;D is a target molecule;Q¹ is a reactive group capable of reacting with a functional group F¹present on a biomolecule;Sp¹ is a spacer moiety;Sp² is a spacer moiety;Sp³ is a spacer moiety;Sp⁴ is a spacer moiety;Z¹ is a connecting group that connects Q¹ or Sp³ to Sp², O or C(O) orN(R¹);Z² is a connecting group that connects D or Sp⁴ to Sp¹, N(R¹), O orC(O); andR¹ is selected from the group consisting of hydrogen, C₁-C₂₄ alkylgroups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups, the C₁-C₂₄alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups,C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groupsoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁-C₄ alkyl groups;or R¹ is D, -[(Sp¹)_(b)-(Z²)_(e)-(Sp⁴)_(i)-D] or-[(Sp²)_(c)-(Z¹)_(d)-(Sp³)_(g)-Q¹], wherein Sp¹, Sp², Sp³, Sp⁴, Z¹, Z²,D, Q1, b, c, d, e, g and i are as defined above.

The invention also relates to a process for the preparation of abioconjugate, the process comprising the step of reacting a reactivegroup Q¹ of a linker-conjugate with a functional group F¹ of abiomolecule, wherein the linker-conjugate is a compound comprising analpha-end and an omega-end, the compound comprising on the alpha-end areactive group Q¹ capable of reacting with a functional group F¹ presenton the biomolecule and on the omega-end a target molecule, the compoundfurther comprising a group according to formula (1) or a salt thereof:

wherein a and R¹ are as defined above, and wherein the group accordingto formula (1), or the salt thereof, is situated in between saidalpha-end and said omega-end of the compound.

More in particular, the invention relates to a process for thepreparation of a bioconjugate, the process comprising the step ofreacting a reactive group Q¹ of a linker-conjugate according to formula(4a) or (4b) as defined above, with a functional group F¹ of abiomolecule. In a preferred embodiment, the invention concerns a processfor the preparation of a bioconjugate via a cycloaddition, such as a(4+2)-cycloaddition (e.g. a Diels-Alder reaction) or a(3+2)-cycloaddition (e.g. a 1,3-dipolar cycloaddition), preferably the1,3-dipolar cycloaddition, more preferably the alkyne-azidecycloaddition, and most preferably wherein Q¹ is or comprises an alkynegroup, such as a cycloalkyne group, and F¹ is an azido group. In afurther preferred embodiment, the invention concerns a process for thepreparation of a bioconjugate, wherein the target molecule ishydrophobic (i.e. weakly soluble in water), most preferably wherein thetarget molecule has a water solubility of at most 0.1% (w/w) in water(20° C. and 100 kPa). In an especially preferred embodiment, theinvention concerns a process for the preparation of a bioconjugate via acycloaddition, preferably a 1,3-dipolar cycloaddition, more preferablythe alkyne-azide cycloaddition, and most preferably wherein Q¹ is orcomprises an alkyne group and F¹ is an azido group, and wherein thetarget molecule is hydrophobic, most preferably wherein the targetmolecule has a water solubility of at most 0.1% (w/w) in water (20° C.and 100 kPa).

The invention further relates to a bioconjugate obtainable by theprocess according to the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 describes the general concept of conjugation of biomolecules: abiomolecule of interest (BOI) containing one or more functional groupsF¹ is incubated with (excess of) a target molecule D (also referred toas molecule of interest or MOI) covalently attached to a reactive groupQ¹ via a specific linker. In the process of bioconjugation, a chemicalreaction between F¹ and Q¹ takes place, thereby forming a bioconjugatecomprising a covalent connection between the BOI and the MOI. The BOImay e.g. be a peptide/protein, a glycan or a nucleic acid.

FIG. 2 shows several structures of derivatives of UDP sugars ofgalactosamine, which may be modified with e.g. a 3-mercaptopropionylgroup (11a), an azidoacetyl group (11b), or an azidodifluoroacetyl group(11c).

FIG. 3 schematically displays how any of the UDP-sugars 11a-c may beattached to a glycoprotein comprising a GlcNAc moiety 12 (e.g. amonoclonal antibody the glycan of which is trimmed by anendoglycosidase) under the action of a galactosyltransferase mutant or aGalNAc-transferase, thereby generating a β-glycosidic 1-4 linkagebetween a GalNAc derivative and GlcNAc (compounds 13a-c, respectively).

FIG. 4 shows how a modified antibody 13a-c may undergo a bioconjugationprocess by means of nucleophilic addition to maleimide (as for 13a,leading to thioether conjugate 14) or upon strain-promoted cycloadditionwith a cyclooctyne reagent (as for 13b or 13c, leading to triazoles 15or 16, respectively).

FIG. 5 shows the structures of several compounds wherein an N-maleimidylreactive group Q¹ is connected to a pyrene group (D) via a linker unit.

FIG. 6 shows the structures of several compounds wherein abicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (also referred to as aBCN group) is connected to benzylamine (D) via a linker unit.

FIG. 7 shows the structures of several compounds wherein abicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (also referred to as aBCN group) or a dibenzoazocyclooctyne reactive group Q¹ (also referredto as a DIBAC group or DBCO group) is connected to pyrene via a linkerunit.

FIG. 8 shows the structures of several compounds wherein abicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (also referred to as aBCN group) is connected to maytansin via a linker unit.

FIG. 9 shows the structures of several compounds wherein abicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (also referred to as aBCN group) is connected to a Val-Cit-PABA-duocarmycin construct via alinker unit.

FIG. 10 shows the structures of several compounds according to theinvention wherein a bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (alsoreferred to as a BCN group) is conjugated to Val-Cit-PABA-Ahx-maytansinvia a linker unit.

FIG. 11 shows the HPLC retention times of compounds 19-23 and 30-38 with0.1% TFA or in buffer pH 7.4.

FIG. 12a shows the conjugation efficiency of BCN-pyrene derivativesconjugated via a sulfamide linker (compound 26) or short PEG linker(compounds 24 or 25) with trastuzumab-N₃ (compound 13b).

FIG. 12b shows the conjugation efficiency of DIBAC-pyrene derivativesconjugated via a sulfamide linker (compound 29) or short PEG linker(compounds 27 or 28) with trastuzumab-N₃ (compound 13b).

FIG. 13a shows the conjugation efficiency of BCN-maytansin derivativesconjugated via a sulfamide linker (compound 33) or short PEG linker(compounds 30-32) with trastuzumab-N₃ (compound 13b).

FIG. 13b shows the conjugation efficiency of BCN-maytansin derivativesconjugated via a sulfamide linker (compounds 33) or short PEG linker(compounds 30-32, with compound 30 overlapping with 33) withtrastuzumab-F₂-GalNAz (compound 13c).

FIG. 14a shows the conjugation efficiency of BCN-duocarmycin derivativesconjugated via a sulfamide linker (compound 35) or short PEG linker(compound 34) with trastuzumab-N₃ (compound 13b).

FIG. 14b shows the conjugation efficiency of BCN-duocarmycin derivativesconjugated via a sulfamide linker (compound 35) or short PEG linker(compound 34) with trastuzumab-F₂-GalNAz (compound 13c).

FIG. 15 shows the general synthetic scheme to convert an alcohol (40)into a carbamoyl sulfamide derivative (42) by consecutive treatment withchlorosulfonyl isocyanate (CSI) and an amine. The carbamoyl sulfamide 42can be converted into an acylsulfamide (44) if the starting alcohol istert-butanol, upon tert-butyl deprotection with acid to give sulfamide43, which can be acylated to give 44.

FIG. 16 shows the synthesis of several linker-conjugates wherein Q¹ isan N-maleimidyl group and D is a pyrene. Compound 18 is according to theinvention, whereas compound 17 is a comparative example.

FIG. 17 shows the synthesis of several linker-conjugates, wherein Q¹ isa bicyclo[6.1.0]non-4-yn-9-yl] group (also referred to as a BCN group)and D is a benzyl group.

FIG. 18 shows the synthesis of a linker-conjugate bearing two sulfamidegroups in the linker, wherein Q¹ is a bicyclo[6.1.0]non-4-yn-9-yl] group(also referred to as a BCN group) and D is a benzyl group.

FIG. 19 shows the synthesis of several linker-conjugates, wherein Q¹ isa bicyclo[6.1.0]non-4-yn-9-yl] group (also referred to as a BCN group)and D is a pyrene group.

FIG. 20 shows the synthesis of several linker-conjugates wherein Q¹ is aheterocycloalkynyl group (DIBAC) and D is a pyrene group.

FIG. 21 shows the synthesis of a linker-conjugate comprising two targetmolecules D, wherein Q¹ is a bicyclo[6.1.0]non-4-yn-9-yl] group (alsoreferred to as a BCN group) and D is a cytotoxin (maytansin).

FIG. 22 shows a representative set of functional groups (F¹) in abiomolecule, either naturally present or introduced by engineering,which upon reaction with reactive group Q¹ lead to connecting group Z³.Connecting group Z³ may also be the result of a reaction between F² andQ². Functional group F¹ may also be artificially introduced (engineered)into a biomolecule at any position of choice.

FIG. 23 shows the extent of aggregation for bioconjugates 36-38according to the invention.

FIG. 24 shows the progress of aggregation over a period of 2 weeks for abioconjugate according to the invention with a sulfamide linker (36conjugated to 13a) vs. a comparative bioconjugate with a PEG linker (30conjugated to 13a), as well as trastuzumab.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The verb “to comprise”, and its conjugations, as used in thisdescription and in the claims is used in its non-limiting sense to meanthat items following the word are included, but items not specificallymentioned are not excluded.

In addition, reference to an element by the indefinite article “a” or“an” does not exclude the possibility that more than one of the elementis present, unless the context clearly requires that there is one andonly one of the elements. The indefinite article “a” or “an” thususually means “at least one”.

The compounds disclosed in this description and in the claims maycomprise one or more asymmetric centres, and different diastereomersand/or enantiomers may exist of the compounds. The description of anycompound in this description and in the claims is meant to include alldiastereomers, and mixtures thereof, unless stated otherwise. Inaddition, the description of any compound in this description and in theclaims is meant to include both the individual enantiomers, as well asany mixture, racemic or otherwise, of the enantiomers, unless statedotherwise. When the structure of a compound is depicted as a specificenantiomer, it is to be understood that the invention of the presentapplication is not limited to that specific enantiomer.

The compounds may occur in different tautomeric forms. The compoundsaccording to the invention are meant to include all tautomeric forms,unless stated otherwise. When the structure of a compound is depicted asa specific tautomer, it is to be understood that the invention of thepresent application is not limited to that specific tautomer.

The compounds disclosed in this description and in the claims mayfurther exist as exo and endo diastereoisomers. Unless stated otherwise,the description of any compound in the description and in the claims ismeant to include both the individual exo and the individual endodiastereoisomers of a compound, as well as mixtures thereof. When thestructure of a compound is depicted as a specific endo or exodiastereomer, it is to be understood that the invention of the presentapplication is not limited to that specific endo or exo diastereomer.

Furthermore, the compounds disclosed in this description and in theclaims may exist as cis and trans isomers. Unless stated otherwise, thedescription of any compound in the description and in the claims ismeant to include both the individual cis and the individual trans isomerof a compound, as well as mixtures thereof. As an example, when thestructure of a compound is depicted as a cis isomer, it is to beunderstood that the corresponding trans isomer or mixtures of the cisand trans isomer are not excluded from the invention of the presentapplication. When the structure of a compound is depicted as a specificcis or trans isomer, it is to be understood that the invention of thepresent application is not limited to that specific cis or trans isomer.

Unsubstituted alkyl groups have the general formula C_(n)H_(2n+1) andmay be linear or branched. Optionally, the alkyl groups are substitutedby one or more substituents further specified in this document. Examplesof alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl,1-hexyl, 1-dodecyl, etc.

A cycloalkyl group is a cyclic alkyl group. Unsubstituted cycloalkylgroups comprise at least three carbon atoms and have the general formulaC_(n)H_(2n−1). Optionally, the cycloalkyl groups are substituted by oneor more substituents further specified in this document. Examples ofcycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl.

An alkenyl group comprises one or more carbon-carbon double bonds, andmay be linear or branched. Unsubstituted alkenyl groups comprising oneC═C double bond have the general formula C_(n)H_(2n−1). Unsubstitutedalkenyl groups comprising two C═C double bonds have the general formulaC_(n)H_(2n−3). An alkenyl group may comprise a terminal carbon-carbondouble bond and/or an internal carbon-carbon double bond. A terminalalkenyl group is an alkenyl group wherein a carbon-carbon double bond islocated at a terminal position of a carbon chain. An alkenyl group mayalso comprise two or more carbon-carbon double bonds. Examples of analkenyl group include ethenyl, propenyl, isopropenyl, t-butenyl,1,3-butadienyl, 1,3-pentadienyl, etc. Unless stated otherwise, analkenyl group may optionally be substituted with one or more,independently selected, substituents as defined below. Unless statedotherwise, an alkenyl group may optionally be interrupted by one or moreheteroatoms independently selected from the group consisting of O, N andS.

An alkynyl group comprises one or more carbon-carbon triple bonds, andmay be linear or branched. Unsubstituted alkynyl groups comprising oneC—C triple bond have the general formula C_(n)H_(2n−3). An alkynyl groupmay comprise a terminal carbon-carbon triple bond and/or an internalcarbon-carbon triple bond. A terminal alkynyl group is an alkynyl groupwherein a carbon-carbon triple bond is located at a terminal position ofa carbon chain. An alkynyl group may also comprise two or morecarbon-carbon triple bonds. Unless stated otherwise, an alkynyl groupmay optionally be substituted with one or more, independently selected,substituents as defined below. Examples of an alkynyl group includeethynyl, propynyl, isopropynyl, t-butynyl, etc. Unless stated otherwise,an alkynyl group may optionally be interrupted by one or moreheteroatoms independently selected from the group consisting of O, N andS.

An aryl group comprises six to twelve carbon atoms and may includemonocyclic and bicyclic structures. Optionally, the aryl group may besubstituted by one or more substituents further specified in thisdocument. Examples of aryl groups are phenyl and naphthyl.

Arylalkyl groups and alkylaryl groups comprise at least seven carbonatoms and may include monocyclic and bicyclic structures. Optionally,the arylalkyl groups and alkylaryl may be substituted by one or moresubstituents further specified in this document. An arylalkyl group isfor example benzyl. An alkylaryl group is for example 4-t-butylphenyl.

Heteroaryl groups comprise at least two carbon atoms (i.e. at least C₂)and one or more heteroatoms N, O, P or S. A heteroaryl group may have amonocyclic or a bicyclic structure. Optionally, the heteroaryl group maybe substituted by one or more substituents further specified in thisdocument. Examples of suitable heteroaryl groups include pyridinyl,quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl,pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl,benzoxazolyl, thienyl, phospholyl and oxazolyl.

Heteroarylalkyl groups and alkylheteroaryl groups comprise at leastthree carbon atoms (i.e. at least C₃) and may include monocyclic andbicyclic structures. Optionally, the heteroaryl groups may besubstituted by one or more substituents further specified in thisdocument.

Where an aryl group is denoted as a (hetero)aryl group, the notation ismeant to include an aryl group and a heteroaryl group. Similarly, analkyl(hetero)aryl group is meant to include an alkylaryl group and aalkylheteroaryl group, and (hetero)arylalkyl is meant to include anarylalkyl group and a heteroarylalkyl group. A C₂-C₂₄ (hetero)aryl groupis thus to be interpreted as including a C₂-C₂₄ heteroaryl group and aC₆-C₂₄ aryl group. Similarly, a C₃-C₂₄ alkyl(hetero)aryl group is meantto include a C₇-C₂₄ alkylaryl group and a C₃-C₂₄ alkylheteroaryl group,and a C₃-C₂₄ (hetero)arylalkyl is meant to include a C₇-C₂₄ arylalkylgroup and a C₃-C₂₄ heteroarylalkyl group.

A cycloalkynyl group is a cyclic alkynyl group. An unsubstitutedcycloalkynyl group comprising one triple bond has the general formulaC_(n)H_(2n−5). Optionally, a cycloalkynyl group is substituted by one ormore substituents further specified in this document. An example of acycloalkynyl group is cyclooctynyl.

A heterocycloalkynyl group is a cycloalkynyl group interrupted byheteroatoms selected from the group of oxygen, nitrogen and sulphur.Optionally, a heterocycloalkynyl group is substituted by one or moresubstituents further specified in this document. An example of aheterocycloalkynyl group is azacyclooctynyl.

A (hetero)aryl group comprises an aryl group and a heteroaryl group. Analkyl(hetero)aryl group comprises an alkylaryl group and analkylheteroaryl group. A (hetero)arylalkyl group comprises a arylalkylgroup and a heteroarylalkyl groups. A (hetero)alkynyl group comprises analkynyl group and a heteroalkynyl group. A (hetero)cycloalkynyl groupcomprises an cycloalkynyl group and a heterocycloalkynyl group.

A (hetero)cycloalkyne compound is herein defined as a compoundcomprising a (hetero)cycloalkynyl group.

Several of the compounds disclosed in this description and in the claimsmay be described as fused (hetero)cycloalkyne compounds, i.e.(hetero)cycloalkyne compounds wherein a second ring structure is fused,i.e. annulated, to the (hetero)cycloalkynyl group. For example in afused (hetero)cyclooctyne compound, a cycloalkyl (e.g. a cyclopropyl) oran arene (e.g. benzene) may be annulated to the (hetero)cyclooctynylgroup. The triple bond of the (hetero)cyclooctynyl group in a fused(hetero)cyclooctyne compound may be located on either one of the threepossible locations, i.e. on the 2, 3 or 4 position of the cyclooctynemoiety (numbering according to “IUPAC Nomenclature of OrganicChemistry”, Rule A31.2). The description of any fused(hetero)cyclooctyne compound in this description and in the claims ismeant to include all three individual regioisomers of the cyclooctynemoiety.

Unless stated otherwise, alkyl groups, cycloalkyl groups, alkenylgroups, alkynyl groups, (hetero)aryl groups, (hetero)arylalkyl groups,alkyl(hetero)aryl groups, alkylene groups, alkenylene groups, alkynylenegroups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylenegroups, (hetero)arylene groups, alkyl(hetero)arylene groups,(hetero)arylalkylene groups, (hetero)arylalkenylene groups,(hetero)arylalkynylene groups, alkenyl groups, alkoxy groups, alkenyloxygroups, (hetero)aryloxy groups, alkynyloxy groups and cycloalkyloxygroups may be substituted with one or more substituents independentlyselected from the group consisting of C₁-C₁₂ alkyl groups, C₂-C₁₂alkenyl groups, C₂-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl groups, C₅-C₁₂cycloalkenyl groups, C₈-C₁₂ cycloalkynyl groups, C₁-C₁₂ alkoxy groups,C₂-C₁₂ alkenyloxy groups, C₂-C₁₂ alkynyloxy groups, C₃-C₁₂ cycloalkyloxygroups, halogens, amino groups, oxo and silyl groups, wherein the silylgroups can be represented by the formula (R²⁰)₃Si—, wherein R²⁰ isindependently selected from the group consisting of C₁-C₁₂ alkyl groups,C₂-C₁₂ alkenyl groups, C₂-C₁₂ alkynyl groups, C₃-C₁₂ cycloalkyl groups,C₁-C₁₂ alkoxy groups, C₂-C₁₂ alkenyloxy groups, C₂-C₁₂ alkynyloxy groupsand C₃-C₁₂ cycloalkyloxy groups, wherein the alkyl groups, alkenylgroups, alkynyl groups, cycloalkyl groups, alkoxy groups, alkenyloxygroups, alkynyloxy groups and cycloalkyloxy groups are optionallysubstituted, the alkyl groups, the alkoxy groups, the cycloalkyl groupsand the cycloalkoxy groups being optionally interrupted by one of morehetero-atoms selected from the group consisting of O, N and S.

The general term “sugar” is herein used to indicate a monosaccharide,for example glucose (Glc), galactose (Gal), mannose (Man) and fucose(Fuc). The term “sugar derivative” is herein used to indicate aderivative of a monosaccharide sugar, i.e. a monosaccharide sugarcomprising substituents and/or functional groups. Examples of a sugarderivative include amino sugars and sugar acids, e.g. glucosamine(GlcNH₂), galactosamine (GalNH₂)N-acetylglucosamine (GlcNAc),N-acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referredto as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid(MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA).

The term “nucleotide” is herein used in its normal scientific meaning.The term “nucleotide” refers to a molecule that is composed of anucleobase, a five-carbon sugar (either ribose or 2-deoxyribose), andone, two or three phosphate groups. Without the phosphate group, thenucleobase and sugar compose a nucleoside. A nucleotide can thus also becalled a nucleoside monophosphate, a nucleoside diphosphate or anucleoside triphosphate. The nucleobase may be adenine, guanine,cytosine, uracil or thymine. Examples of a nucleotide include uridinediphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate(TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP).

The term “protein” is herein used in its normal scientific meaning.Herein, polypeptides comprising about 10 or more amino acids areconsidered proteins. A protein may comprise natural, but also unnaturalamino acids.

The term “glycoprotein” is herein used in its normal scientific meaningand refers to a protein comprising one or more monosaccharide oroligosaccharide chains (“glycans”) covalently bonded to the protein. Aglycan may be attached to a hydroxyl group on the protein(O-linked-glycan), e.g. to the hydroxyl group of serine, threonine,tyrosine, hydroxylysine or hydroxyproline, or to an amide function onthe protein (N-glycoprotein), e.g. asparagine or arginine, or to acarbon on the protein (C-glycoprotein), e.g. tryptophan. A glycoproteinmay comprise more than one glycan, may comprise a combination of one ormore monosaccharide and one or more oligosaccharide glycans, and maycomprise a combination of N-linked, O-linked and C-linked glycans. It isestimated that more than 50% of all proteins have some form ofglycosylation and therefore qualify as glycoprotein. Examples ofglycoproteins include PSMA (prostate-specific membrane antigen), CAL(candida antartica lipase), gp41, gp120, EPO (erythropoietin),antifreeze protein and antibodies.

The term “glycan” is herein used in its normal scientific meaning andrefers to a monosaccharide or oligosaccharide chain that is linked to aprotein. The term glycan thus refers to the carbohydrate-part of aglycoprotein. The glycan is attached to a protein via the C-1 carbon ofone sugar, which may be without further substitution (monosaccharide) ormay be further substituted at one or more of its hydroxyl groups(oligosaccharide). A naturally occurring glycan typically comprises 1 toabout 10 saccharide moieties. However, when a longer saccharide chain islinked to a protein, said saccharide chain is herein also considered aglycan. A glycan of a glycoprotein may be a monosaccharide. Typically, amonosaccharide glycan of a glycoprotein consists of a singleN-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose(Fuc) covalently attached to the protein. A glycan may also be anoligosaccharide. An oligosaccharide chain of a glycoprotein may belinear or branched. In an oligosaccharide, the sugar that is directlyattached to the protein is called the core sugar. In an oligosaccharide,a sugar that is not directly attached to the protein and is attached toat least two other sugars is called an internal sugar. In anoligosaccharide, a sugar that is not directly attached to the proteinbut to a single other sugar, i.e. carrying no further sugar substituentsat one or more of its other hydroxyl groups, is called the terminalsugar. For the avoidance of doubt, there may exist multiple terminalsugars in an oligosaccharide of a glycoprotein, but only one core sugar.A glycan may be an O-linked glycan, an N-linked glycan or a C-linkedglycan. In an O-linked glycan a monosaccharide or oligosaccharide glycanis bonded to an O-atom in an amino acid of the protein, typically via ahydroxyl group of serine (Ser) or threonine (Thr). In an N-linked glycana monosaccharide or oligosaccharide glycan is bonded to the protein viaan N-atom in an amino acid of the protein, typically via an amidenitrogen in the side chain of asparagine (Asn) or arginine (Arg). In aC-linked glycan a monosaccharide or oligosaccharide glycan is bonded toa C-atom in an amino acid of the protein, typically to a C-atom oftryptophan (Trp).

The term “antibody” is herein used in its normal scientific meaning. Anantibody is a protein generated by the immune system that is capable ofrecognizing and binding to a specific antigen. An antibody is an exampleof a glycoprotein. The term antibody herein is used in its broadestsense and specifically includes monoclonal antibodies, polyclonalantibodies, dimers, multimers, multispecific antibodies (e.g. bispecificantibodies), antibody fragments, and double and single chain antibodies.The term “antibody” is herein also meant to include human antibodies,humanized antibodies, chimeric antibodies and antibodies specificallybinding cancer antigen. The term “antibody” is meant to include wholeantibodies, but also fragments of an antibody, for example an antibodyFab fragment, F(ab′)₂, Fv fragment or Fc fragment from a cleavedantibody, a scFv-Fc fragment, a minibody, a diabody or a scFv.Furthermore, the term includes genetically engineered antibodies andderivatives of an antibody. Antibodies, fragments of antibodies andgenetically engineered antibodies may be obtained by methods that areknown in the art. Typical examples of antibodies include, amongstothers, abciximab, rituximab, basiliximab, palivizumab, infliximab,trastuzumab, alemtuzumab, adalimumab, tositumomab-I131, cetuximab,ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab, ranibizumab,panitumumab, eculizumab, certolizumab pegol, golimumab, canakinumab,catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab,ipilimumab and brentuximab.

A linker is herein defined as a moiety that connects two or moreelements of a compound. For example in a bioconjugate, a biomolecule anda target molecule are covalently connected to each other via a linker;in a linker-conjugate a reactive group Q¹ is covalently connected to atarget molecule via a linker; in a linker-construct a reactive group Q¹is covalently connected to a reactive group Q² via a linker. A linkermay comprise one or more spacer-moieties.

A spacer-moiety is herein defined as a moiety that spaces (i.e. providesdistance between) and covalently links together two (or more) parts of alinker. The linker may be part of e.g. a linker-construct, alinker-conjugate or a bioconjugate, as defined below.

A linker-construct is herein defined as a compound wherein a reactivegroup Q¹ is covalently connected to a reactive group Q² via a linker. Alinker-construct comprises a reactive group Q¹ capable of reacting witha reactive group present on a biomolecule, and a reactive group Q²capable of reacting with a reactive group present on a target molecule.Q¹ and Q² may be the same, or different. A linker-construct may alsocomprise more than one reactive group Q¹ and/or more than one reactivegroup Q². A linker-construct may also be denoted as Q¹-Sp-Q², wherein Q¹is a reactive group capable of reacting with a reactive group F¹ presenton a biomolecule, Q² is a reactive group capable of reacting with areactive group F² present on a target molecule and Sp is a spacermoiety. When a linker-construct comprises more than one reactive groupQ¹ and/or more than one reactive group Q², the linker-construct may bedenoted as (Q¹)_(y)-Sp-(Q²)_(z), wherein y is an integer in the range of1 to 10 and z is an integer in the range of 1 to 10. Preferably, y is 1,2, 3 or 4, more preferably y is 1 or 2 and most preferably, y is 1.Preferably, z is 1, 2, 3, 4, 5 or 6, more preferably z is 1, 2, 3 or 4,even more preferably z is 1, 2 or 3, yet even more preferably z is 1 or2 and most preferably z is 1. More preferably, y is 1 or 2, preferably1, and z is 1, 2, 3 or 4, yet even more preferably y is 1 or 2,preferably 1, and z is 1, 2 or 3, yet even more preferably y is 1 or 2,preferably 1, and z is 1 or 2, and most preferably y is 1 and z is 1.

A linker-conjugate is herein defined as a compound wherein a targetmolecule is covalently connected to a reactive group Q¹, via a linker. Alinker-conjugate may be obtained via reaction of a reactive group Q²present on a linker-construct with a reactive group present on a targetmolecule. A linker-conjugate comprises a reactive group Q¹ that iscapable of reacting with a reactive group present on a biomolecule. Alinker-conjugate may comprise one or more spacer moieties. Alinker-conjugate may comprise more than one reactive groups Q¹ and/ormore than one target molecules.

A bioconjugate is herein defined as a compound wherein a biomolecule iscovalently connected to a target molecule via a linker. A bioconjugatecomprises one or more biomolecules and/or one or more target molecules.The linker may comprise one or more spacer moieties.

When a compound is herein referred to as a compound comprising analpha-end and an omega-end, said compound comprises two (or more) ends,the first end being referred to as the alpha-end and the second endbeing referred to as the omega-end. Said compound may comprise more thantwo ends, i.e. a third, fourth etc. end may be present in the compound.

A biomolecule is herein defined as any molecule that can be isolatedfrom Nature or any molecule composed of smaller molecular buildingblocks that are the constituents of macromolecular structures derivedfrom Nature, in particular nucleic acids, proteins, glycans and lipids.Examples of a biomolecule include an enzyme, a (non-catalytic) protein,a polypeptide, a peptide, an amino acid, an oligonucleotide, amonosaccharide, an oligosaccharide, a polysaccharide, a glycan, a lipidand a hormone.

A target molecule, also referred to as a molecule of interest (MOD, isherein defined as molecular structure possessing a desired property thatis imparted onto the biomolecule upon conjugation.

The term “salt thereof” means a compound formed when an acidic proton,typically a proton of an acid, is replaced by a cation, such as a metalcation or an organic cation and the like. Where applicable, the salt isa pharmaceutically acceptable salt, although this is not required forsalts that are not intended for administration to a patient. Forexample, in a salt of a compound the compound may be protonated by aninorganic or organic acid to form a cation, with the conjugate base ofthe inorganic or organic acid as the anionic component of the salt.

The term “pharmaceutically accepted” salt means a salt that isacceptable for administration to a patient, such as a mammal (salts withcounterions having acceptable mammalian safety for a given dosageregime). Such salts may be derived from pharmaceutically acceptableinorganic or organic bases and from pharmaceutically acceptableinorganic or organic acids. “Pharmaceutically acceptable salt” refers topharmaceutically acceptable salts of a compound, which salts are derivedfrom a variety of organic and inorganic counter ions known in the artand include, for example, sodium, potassium, calcium, magnesium,ammonium, tetraalkylammonium, etc., and when the molecule contains abasic functionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, formate, tartrate, besylate, mesylate,acetate, maleate, oxalate, etc.

Linker-Conjugate

Herein, a sulfamide linker and conjugates of said sulfamide linker aredisclosed. The term “sulfamide linker” refers to a linker comprising asulfamide group, more particularly an acylsulfamide group[—C(O)—N(H)—S(O)₂—N(R¹)—] and/or a carbamoyl sulfamide group[—O—C(O)—N(H)—S(O)₂—N(R¹)—].

The present invention relates to the use of a sulfamide linker accordingto the invention in a process for the preparation of a bioconjugate. Theinvention further relates to a process for the preparation of abioconjugate and to a bioconjugate obtainable by said process. Saidprocess for the preparation of a bioconjugate is described in moredetail below.

In a first aspect, the present invention relates to a compoundcomprising an alpha-end and an omega-end, the compound comprising on thealpha-end a reactive group Q¹ capable of reacting with a functionalgroup F¹ present on a biomolecule and on the omega-end a target moleculeD, the compound further comprising a group according to formula (1) or asalt thereof:

wherein:

-   -   a is 0 or 1; and    -   R¹ is selected from the group consisting of hydrogen, C₁-C₂₄        alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl        groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups, the C₁-C₂₄ alkyl groups, C₃-C₂₄        cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups, or R¹ is a target molecule D, wherein the        target molecule is optionally connected to N via a spacer        moiety;        and wherein the group according to formula (1), or the salt        thereof, is situated in between said alpha-end and said        omega-end of the compound.

Said compound is also referred to as a linker-conjugate. Alinker-conjugate is herein defined as a compound wherein a targetmolecule is covalently connected to a reactive group Q¹, via a linker.The compound comprises an alpha-end and an omega-end, in other words,the compound comprises a first end and a second end. The first end ofthe compound may also be referred to as an alpha-end, and the second endmay also be referred to as an omega-end of the compound. The terms“alpha-end” and “omega-end” are known to a person skilled in the art.The invention thus also relates to a compound comprising a first end anda second end, the compound comprising on the first end a reactive groupQ¹ capable of reacting with a functional group F¹ present on abiomolecule and on the second end a target molecule D, the compoundfurther comprising a group according to formula (1) or a salt thereof,wherein the group according to formula (1), or the salt thereof, issituated in between said first end and said second end of the compound,and wherein the group according to formula (1) is as defined above.

In the compound according to invention, the group according to formula(1), or the salt thereof, is situated in between said alpha-end and saidomega-end of the compound. Reactive group Q¹ is covalently bonded to thealpha-end of the compound, and target molecule D is covalently bonded toan omega-end of the compound.

The compound according to the invention may also be referred to as alinker-conjugate. In the linker-conjugate according to the invention, atarget molecule D is covalently attached to a reactive group Q¹ via alinker, and said linker comprises a group according to formula (1), or asalt thereof, as defined above.

When the linker-conjugate according to the invention comprises a salt ofthe group according to formula (1), the salt is preferably apharmaceutically acceptable salt.

The linker-conjugate according to the invention may comprise more thanone target molecule D. Consequently, the linker-conjugate may thuscomprise more than one omega-end, e.g. a second (third, fourth, fifth,etc.) omega-end, the second (third, fourth, fifth, etc.) omega-end maybe covalently attached to a target molecule. Similarly, thelinker-conjugate may comprise more than one reactive group Q¹, i.e. thelinker-conjugate may comprise more than one alpha-end. When more thanone reactive group Q¹ is present the groups Q¹ may be the same ordifferent, and when more than one target molecule D is present thetarget molecules D may be the same or different.

The linker-conjugate according to the invention may therefore also bedenoted as (Q¹)_(y)-Sp-(D)_(z), wherein y is an integer in the range of1 to 10 and z is an integer in the range of 1 to 10.

The invention thus also relates to a compound according to the formula:(Q¹)_(y)-Sp-(D)_(z),wherein:y is an integer in the range of 1 to 10;z is an integer in the range of 1 to 10;Q¹ is a reactive group capable of reacting with a functional group F¹present on a biomolecule;D is a target molecule;Sp is a spacer moiety, wherein a spacer moiety is defined as a moietythat spaces (i.e. provides a certain distance between) and covalentlylinks reactive group Q¹ and target molecule D; andwherein said spacer moiety comprises a group according to Formula (1) ora salt thereof, wherein the group according to Formula (1) is as definedabove.

Preferably, y is 1, 2, 3 or 4, more preferably y is 1 or 2 and mostpreferably, y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferablyz is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, yet even morepreferably z is 1 or 2 and most preferably z is 1. More preferably, y is1 or 2, preferably 1, and z is 1, 2, 3 or 4, yet even more preferably yis 1 or 2, preferably 1, and z is 1, 2 or 3, yet even more preferably yis 1 or 2, preferably 1, and z is 1 or 2, and most preferably y is 1 andz is 1. In a preferred embodiment, the linker-conjugate is according tothe formula Q¹-Sp-(D)₄, Q¹-Sp-(D)₃, Q¹-Sp-(D)₂ or Q¹-Sp-D.

The linker-conjugate according to the invention comprises a groupaccording to formula (1) as defined above, or a salt thereof. In apreferred embodiment, the linker-conjugate according to the inventioncomprises a group according to formula (1) wherein a is 0, or a saltthereof. In this embodiment, the compound thus comprises a groupaccording to formula (2) or a salt thereof:

wherein R¹ is as defined above.

In another preferred embodiment, the linker-conjugate according to theinvention comprises a group according to formula (1) wherein a is 1, ora salt thereof. In this embodiment, the linker-conjugate thus comprisesa group according to formula (3) or a salt thereof:

wherein R¹ is as defined above.

In the groups according to formula (1), (2) and (3), R¹ is selected fromthe group consisting of hydrogen, C₁-C₂₄ alkyl groups, C₃-C₂₄ cycloalkylgroups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄ alkyl(hetero)aryl groups andC₃-C₂₄ (hetero)arylalkyl groups, the C₁-C₂₄ alkyl groups, C₃-C₂₄cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄ alkyl(hetero)arylgroups and C₃-C₂₄ (hetero)arylalkyl groups optionally substituted andoptionally interrupted by one or more heteroatoms selected from O, S andNR³ wherein R³ is independently selected from the group consisting ofhydrogen and C₁-C₄ alkyl groups, or R¹ is a target molecule D, whereinthe target molecule is optionally connected to N via a spacer moiety;

In a preferred embodiment, R¹ is hydrogen or a C₁-C₂₀ alkyl group, morepreferably R¹ is hydrogen or a C₁-C₁₆ alkyl group, even more preferablyR¹ is hydrogen or a C₁-C₁₀ alkyl group, wherein the alkyl group isoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³, preferably O, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups. In a preferred embodiment, R¹ is hydrogen. In anotherpreferred embodiment, R¹ is a C₁-C₂₀ alkyl group, more preferably aC₁-C₁₆ alkyl group, even more preferably a C₁-C₁₀ alkyl group, whereinthe alkyl group is optionally interrupted by one or more O-atoms, andwherein the alkyl group is optionally substituted with an —OH group,preferably a terminal —OH group. In this embodiment it is furtherpreferred that R¹ is a (poly)ethyleneglycol chain comprising a terminal—OH group. In another preferred embodiment, R¹ is selected from thegroup consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl and t-butyl, more preferably from the group consistingof hydrogen, methyl, ethyl, n-propyl and i-propyl, and even morepreferably from the group consisting of hydrogen, methyl and ethyl. Yeteven more preferably R¹ is hydrogen or methyl, and most preferably R¹ ishydrogen.

In another preferred embodiment, R¹ is a target molecule D. Optionally,the target molecule D is connected to N via one or more spacer-moieties.The spacer-moiety, if present, is defined as a moiety that spaces, i.e.provides a certain distance between, and covalently links targetmolecule D and N. The target molecule D and preferred embodimentsthereof are described in more detail below.

When the linker-conjugate according to the invention comprises two ormore target molecules D, the target molecules D may differ from eachother.

In a preferred embodiment of the linker-conjugate according to theinvention, the target molecule is selected from the group consisting ofan active substance, a reporter molecule, a polymer, a solid surface, ahydrogel, a nanoparticle, a microparticle and a biomolecule.

The inventors found that the use of a sulfamide linker according to theinvention improves the solubility of the linker-conjugate, which in turnsignificantly improves the efficacy of the conjugation. Usingconventional linkers, effective conjugation is often hampered by therelatively low solubility of the linker-conjugate in aqueous media,especially when a relative water-insoluble or hydrophobic targetmolecule is used. In a particularly preferred embodiment, the targetmolecule in its unconjugated form is hydrophobic, typically having awater solubility of at most 1% (w/w), preferably at most 0.1% (w/w),most preferably at most 0.01% (w/w), determined at 20° C. and 100 kPa.Even such water-insoluble target molecules are effectively subjected toconjugation when functionalized with a sulfamide linker according to theinvention.

Herein, the “unconjugated form” refers to the target molecule not beingfunctionalized with or conjugated to the linker according to theinvention. Such unconjugated forms of target molecules are known to theskilled person.

The term “active substance” herein relates to a pharmacological and/orbiological substance, i.e. a substance that is biologically and/orpharmaceutically active, for example a drug, a prodrug, a diagnosticagent, a protein, a peptide, a polypeptide, a peptide tag, an aminoacid, a glycan, a lipid, a vitamin, a steroid, a nucleotide, anucleoside, a polynucleotide, RNA or DNA. Examples of peptide tagsinclude cell-penetrating peptides like human lactoferrin orpolyarginine. An example of a glycan is oligomannose. An example of anamino acid is lysine.

When the target molecule is an active substance, the active substance ispreferably selected from the group consisting of drugs and prodrugs.More preferably, the active substance is selected from the groupconsisting of pharmaceutically active compounds, in particular low tomedium molecular weight compounds (e.g. about 200 to about 2500 Da,preferably about 300 to about 1750 Da). In a further preferredembodiment, the active substance is selected from the group consistingof cytotoxins, antiviral agents, antibacterials agents, peptides andoligonucleotides. Examples of cytotoxins include colchicine, vincaalkaloids, anthracyclines, camptothecins, doxorubicin, daunorubicin,taxanes, calicheamycins, tubulysins, irinotecans, an inhibitory peptide,amanitin, deBouganin, duocarmycins, maytansines, auristatins orpyrrolobenzodiazepines (PBDs). In view of their poor water solubility,preferred active substances include vinca alkaloids, anthracyclines,camptothecins, taxanes, tubulysins, amanitin, duocarmycins, maytansines,auristatins and pyrrolobenzodiazepines, in particular vinca alkaloids,anthracyclines, camptothecins, taxanes, tubulysins, amanitin,maytansines and auristatins.

The term “reporter molecule” herein refers to a molecule whose presenceis readily detected, for example a diagnostic agent, a dye, afluorophore, a radioactive isotope label, a contrast agent, a magneticresonance imaging agent or a mass label.

A wide variety of fluorophores, also referred to as fluorescent probes,is known to a person skilled in the art. Several fluorophores aredescribed in more detail in e.g. G. T. Hermanson, “BioconjugateTechniques”, Elsevier, 3^(rd) Ed. 2013, Chapter 10: “Fluorescentprobes”, p. 395-463, incorporated by reference. Examples of afluorophore include all kinds of Alexa Fluor (e.g. Alexa Fluor 555),cyanine dyes (e.g. Cy3 or Cy5) and cyanine dye derivatives, coumarinderivatives, fluorescein and fluorescein derivatives, rhodamine andrhodamine derivatives, boron dipyrromethene derivatives, pyrenederivatives, naphthalimide derivatives, phycobiliprotein derivatives(e.g. allophycocyanin), chromomycin, lanthanide chelates and quantum dotnanocrystals. In view of their poor water solubility, preferredfluorophores include cyanine dyes, coumarin derivatives, fluorescein andderivatives thereof, pyrene derivatives, naphthalimide derivatives,chromomycin, lanthanide chelates and quantum dot nanocrystals, inparticular coumarin derivatives, fluorescein, pyrene derivatives andchromomycin.

Examples of a radioactive isotope label include ^(99m)Tc, ¹¹¹In,^(114m)In, ¹¹⁵In, ¹⁸F, ¹⁴C, ⁶⁴Cu, ¹³¹I, ¹²⁵I, ¹²³I, ²¹²Bi, ⁸⁸Y, ⁹⁰Y,⁶⁷Cu, ¹⁸⁶Rh, ¹⁸⁸Rh, ⁶⁶Ga, ⁶⁷Ga and ¹⁰B, which is optionally connectedvia a chelating moiety such as e.g. DTPA (diethylenetriaminepentaaceticanhydride), DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraaceticacid), NOTA (1,4,7-triazacyclononane N,N′,N″-triacetic acid), TETA(1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid), DTTA(N¹-(p-isothiocyanatobenzyl)-diethylenetriamine-N¹,N²,N³,N³-tetraaceticacid), deferoxamine or DFA(N′-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxy-amino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide)or HYNIC (hydrazino-nicotinamide). Isotopic labelling techniques areknown to a person skilled in the art, and are described in more detailin e.g. G. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3^(rd) Ed.2013, Chapter 12: “Isotopic labelling techniques”, p. 507-534,incorporated by reference.

Polymers suitable for use as a target molecule D in the compoundaccording to the invention are known to a person skilled in the art, andseveral examples are described in more detail in e.g. G. T. Hermanson,“Bioconjugate Techniques”, Elsevier, 3^(rd) Ed. 2013, Chapter 18:“PEGylation and synthetic polymer modification”, p. 787-838,incorporated by reference. When target molecule D is a polymer, targetmolecule D is preferably independently selected from the groupconsisting of a poly(ethyleneglycol) (PEG), a polyethylene oxide (PEO),a polypropylene glycol (PPG), a polypropylene oxide (PPO), a1,x-diaminoalkane polymer (wherein x is the number of carbon atoms inthe alkane, and preferably x is an integer in the range of 2 to 200,preferably 2 to 10), a (poly)ethylene glycol diamine (e.g.1,8-diamino-3,6-dioxaoctane and equivalents comprising longer ethyleneglycol chains), a polysaccharide (e.g. dextran), a poly(amino acid)(e.g. a poly(L-lysine)) and a poly(vinyl alcohol). In view of their poorwater solubility, preferred polymers include a 1,x-diaminoalkane polymerand poly(vinyl alcohol).

Solid surfaces suitable for use as a target molecule D are known to aperson skilled in the art. A solid surface is for example a functionalsurface (e.g. a surface of a nanomaterial, a carbon nanotube, afullerene or a virus capsid), a metal surface (e.g. a titanium, gold,silver, copper, nickel, tin, rhodium or zinc surface), a metal alloysurface (wherein the alloy is from e.g. aluminium, bismuth, chromium,cobalt, copper, gallium, gold, indium, iron, lead, magnesium, mercury,nickel, potassium, plutonium, rhodium, scandium, silver, sodium,titanium, tin, uranium, zinc and/or zirconium), a polymer surface(wherein the polymer is e.g. polystyrene, polyvinylchloride,polyethylene, polypropylene, poly(dimethylsiloxane) orpolymethylmethacrylate, polyacrylamide), a glass surface, a siliconesurface, a chromatography support surface (wherein the chromatographysupport is e.g. a silica support, an agarose support, a cellulosesupport or an alumina support), etc. When target molecule D is a solidsurface, it is preferred that D is independently selected from the groupconsisting of a functional surface or a polymer surface.

Hydrogels are known to the person skilled in the art. Hydrogels arewater-swollen networks, formed by cross-links between the polymericconstituents. See for example A. S. Hoffman, Adv. Drug Delivery Rev.2012, 64, 18, incorporated by reference. When the target molecule is ahydrogel, it is preferred that the hydrogel is composed ofpoly(ethylene)glycol (PEG) as the polymeric basis.

Micro- and nanoparticles suitable for use as a target molecule D areknown to a person skilled in the art. A variety of suitable micro- andnanoparticles is described in e.g. G. T. Hermanson, “BioconjugateTechniques”, Elsevier, 3^(rd) Ed. 2013, Chapter 14: “Microparticles andnanoparticles”, p. 549-587, incorporated by reference. The micro- ornanoparticles may be of any shape, e.g. spheres, rods, tubes, cubes,triangles and cones. Preferably, the micro- or nanoparticles are of aspherical shape. The chemical composition of the micro- andnanoparticles may vary. When target molecule D is a micro- or ananoparticle, the micro- or nanoparticle is for example a polymericmicro- or nanoparticle, a silica micro- or nanoparticle or a gold micro-or nanoparticle. When the particle is a polymeric micro- ornanoparticle, the polymer is preferably polystyrene or a copolymer ofstyrene (e.g. a copolymer of styrene and divinylbenzene, butadiene,acrylate and/or vinyltoluene), polymethylmethacrylate (PMMA),polyvinyltoluene, poly(hydroxyethyl methacrylate (pHEMA) orpoly(ethylene glycol dimethacrylate/2-hydroxyethylmetacrylae)[poly(EDGMA/HEMA)]. Optionally, the surface of the micro- ornanoparticles is modified, e.g. with detergents, by graft polymerizationof secondary polymers or by covalent attachment of another polymer or ofspacer moieties, etc.

Target molecule D may also be a biomolecule. Biomolecules, and preferredembodiments thereof, are described in more detail below. When targetmolecule D is a biomolecule, it is preferred that the biomolecule isselected from the group consisting of proteins (including glycoproteinsand antibodies), polypeptides, peptides, glycans, lipids, nucleic acids,oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones,amino acids and monosaccharides.

The linker-conjugate according to the invention comprises a reactivegroup Q¹ that is capable of reacting with a functional group F¹ presenton a biomolecule. Functional groups are known to a person skilled in theart and may be defined as any molecular entity that imparts a specificproperty onto the molecule harbouring it. For example, a functionalgroup in a biomolecule may constitute an amino group, a thiol group, acarboxylic acid, an alcohol group, a carbonyl group, a phosphate group,or an aromatic group. The functional group in the biomolecule may benaturally present or may be placed in the biomolecule by a specifictechnique, for example a (bio)chemical or a genetic technique. Thefunctional group that is placed in the biomolecule may be a functionalgroup that is naturally present in nature, or may be a functional groupthat is prepared by chemical synthesis, for example an azide, a terminalalkyne or a phosphine moiety. Herein, the term “reactive group” mayrefer to a certain group that comprises a functional group, but also toa functional group itself. For example, a cyclooctynyl group is areactive group comprising a functional group, namely a C—C triple bond.Similarly, an N-maleimidyl group is a reactive group, comprising a C═Cdouble bond as a functional group. However, a functional group, forexample an azido functional group, a thiol functional group or an aminofunctional group, may herein also be referred to as a reactive group.

The linker-conjugate may comprise more than one reactive group Q¹. Whenthe linker-conjugate comprises two or more reactive groups Q¹, thereactive groups Q¹ may differ from each other. Preferably, thelinker-conjugate comprises one reactive group Q¹.

Reactive group Q¹ that is present in the linker-conjugate, is able toreact with a functional group F¹ that is present in a biomolecule. Inother words, reactive group Q¹ needs to be complementary to a functionalgroup F¹ present in a biomolecule. Herein, a reactive group is denotedas “complementary” to a functional group when said reactive group reactswith said functional group selectively, optionally in the presence ofother functional groups. Complementary reactive and functional groupsare known to a person skilled in the art, and are described in moredetail below.

In a preferred embodiment, reactive group Q¹ is selected from the groupconsisting of, optionally substituted, N-maleimidyl groups, halogenatedN-alkylamido groups, sulfonyloxy N-alkylamido groups, ester groups,carbonate groups, sulfonyl halide groups, thiol groups or derivativesthereof, alkenyl groups, alkynyl groups, (hetero)cycloalkynyl groups,bicyclo[6.1.0]non-4-yn-9-yl] groups, cycloalkenyl groups, tetrazinylgroups, azido groups, phosphine groups, nitrile oxide groups, nitronegroups, nitrile imine groups, diazo groups, ketone groups,(O-alkyl)hydroxylamino groups, hydrazine groups, halogenatedN-maleimidyl groups, 1,1-bis(sulfonylmethyl)-methylcarbonyl groups orelimination derivatives thereof, carbonyl halide groups, allenamidegroups, 1,2-quinone groups or triazine groups.

In a preferred embodiment, Q¹ is an N-maleimidyl group. When Q¹ is anN-maleimidyl group, Q¹ is preferably unsubstituted. Q¹ is thuspreferably according to formula (9a), as shown below.

In another preferred embodiment, Q¹ is a halogenated N-alkylamido group.When Q¹ is a halogenated N-alkylamido group, it is preferred that Q¹ isaccording to formula (9b), as shown below, wherein k is an integer inthe range of 1 to 10 and R⁴ is selected from the group consisting of—Cl, —Br and —I. Preferably k is 1, 2, 3 or 4, more preferably k is 1 or2 and most preferably k is 1. Preferably, R⁴ is —I or —Br. Morepreferably, k is 1 or 2 and R⁴ is —I or —Br, and most preferably k is 1and R⁴ is —I or Br.

In another preferred embodiment, Q¹ is a sulfonyloxy N-alkylamido group.When Q¹ is a sulfonyloxy N-alkylamido group, it is preferred that Q¹ isaccording to formula (9b), as shown below, wherein k is an integer inthe range of 1 to 10 and R⁴ is selected from the group consisting of—O-mesyl, —O-phenylsulfonyl and —O-tosyl. Preferably k is 1, 2, 3 or 4,more preferably k is 1 or 2, even more preferably k is 1. Mostpreferably k is 1 and R⁴ is selected from the group consisting of—O-mesyl, —O-phenylsulfonyl and —O-tosyl.

In another preferred embodiment, Q¹ is an ester group. When Q¹ is anester group, it is preferred that the ester group is an activated estergroup. Activated ester groups are known to the person skilled in theart. An activated ester group is herein defined as an ester groupcomprising a good leaving group, wherein the ester carbonyl group isbonded to said good leaving group. Good leaving groups are known to theperson skilled in the art. It is further preferred that the activatedester is according to formula (9c), as shown below, wherein R⁵ isselected from the group consisting of —N— succinimidyl (NHS),—N-sulfo-succinimidyl (sulfo-NHS), -(4-nitrophenyl), -pentafluorophenylor -tetrafluorophenyl (TFP).

In another preferred embodiment, Q¹ is a carbonate group. When Q¹ is acarbonate group, it is preferred that the carbonate group is anactivated carbonate group. Activated carbonate groups are known to aperson skilled in the art. An activated carbonate group is hereindefined as a carbonate group comprising a good leaving group, whereinthe carbonate carbonyl group is bonded to said good leaving group. It isfurther preferred that the carbonate group is according to formula (9d),as shown below, wherein R⁷ is selected from the group consisting of—N-succinimidyl, —N-sulfo-succinimidyl, -(4-nitrophenyl),-pentafluorophenyl or -tetrafluorophenyl.

In another preferred embodiment, Q¹ is a sulfonyl halide group accordingto formula (9e) as shown below, wherein X is selected from the groupconsisting of F, Cl, Br and I. Preferably X is Cl or Br, more preferablyCl.

In another preferred embodiment, Q¹ is a thiol group (9f), or aderivative or a precursor of a thiol group. A thiol group may also bereferred to as a mercapto group. When Q¹ is a derivative or a precursorof a thiol group, the thiol derivative is preferably according toformula (9g), (9h) or (9zb) as shown below, wherein R⁸ is an, optionallysubstituted, C₁-C₁₂ alkyl group or a C₂-C₁₂ (hetero)aryl group, V is 0or S and R¹⁶ is an, optionally substituted, C₁-C₁₂ alkyl group. Morepreferably R⁸ is an, optionally substituted, C₁-C₆ alkyl group or aC₂-C₆ (hetero)aryl group, and even more preferably R⁸ is methyl, ethyl,n-propyl, i-propyl, n-butyl, s-butyl, t-butyl or phenyl. Even morepreferably, R⁸ is methyl or phenyl, most preferably methyl. Morepreferably R¹⁶ is an optionally substituted C₁-C₆ alkyl group, and evenmore preferably R¹⁶ is methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl or t-butyl, most preferably methyl. When Q¹ is athiol-derivative according to formula (9g) or (9zb), and Q¹ is reactedwith a reactive group F¹ on a biomolecule, said thiol-derivative isconverted to a thiol group during the process. When Q¹ is according toformula (9h), Q¹ is —SC(O)OR⁸ or —SC(S)OR⁸, preferably SC(O)OR⁸, whereinR⁸, and preferred embodiments thereof, are as defined above.

In another preferred embodiment, Q¹ is an alkenyl group, wherein thealkenyl group is linear or branched, and wherein the alkenyl group isoptionally substituted. The alkenyl group may be a terminal or aninternal alkenyl group. The alkenyl group may comprise more than one C═Cdouble bond, and if so, preferably comprises two C—C double bonds. Whenthe alkenyl group is a dienyl group, it is further preferred that thetwo C═C double bonds are separated by one C—C single bond (i.e. it ispreferred that the dienyl group is a conjugated dienyl group).Preferably said alkenyl group is a C₂-C₂₄ alkenyl group, more preferablya C₂-C₁₂ alkenyl group, and even more preferably a C₂-C₆ alkenyl group.It is further preferred that the alkenyl group is a terminal alkenylgroup. More preferably, the alkenyl group is according to formula (9i)as shown below, wherein 1 is an integer in the range of 0 to 10,preferably in the range of 0 to 6, and p is an integer in the range of 0to 10, preferably 0 to 6. More preferably, 1 is 0, 1, 2, 3 or 4, morepreferably 1 is 0, 1 or 2 and most preferably 1 is 0 or 1. Morepreferably, p is 0, 1, 2, 3 or 4, more preferably p is 0, 1 or 2 andmost preferably p is 0 or 1. It is particularly preferred that p is 0and 1 is 0 or 1, or that p is 1 and 1 is 0 or 1.

In another preferred embodiment, Q¹ is an alkynyl group, wherein thealkynyl group is linear or branched, and wherein the alkynyl group isoptionally substituted. The alkynyl group may be a terminal or aninternal alkynyl group. Preferably said alkynyl group is a C₂-C₂₄alkynyl group, more preferably a C₂-C₁₂ alkynyl group, and even morepreferably a C₂-C₆ alkynyl group. It is further preferred that thealkynyl group is a terminal alkynyl group. More preferably, the alkynylgroup is according to formula (9j) as shown below, wherein 1 is aninteger in the range of 0 to 10, preferably in the range of 0 to 6. Morepreferably, 1 is 0, 1, 2, 3 or 4, more preferably 1 is 0, 1 or 2 andmost preferably 1 is 0 or 1.

In another preferred embodiment, Q¹ is a cycloalkenyl group. Thecycloalkenyl group is optionally substituted. Preferably saidcycloalkenyl group is a C₃-C₂₄ cycloalkenyl group, more preferably aC₃-C₁₂ cycloalkenyl group, and even more preferably a C₃-C₈ cycloalkenylgroup. In a preferred embodiment, the cycloalkenyl group is atrans-cycloalkenyl group, more preferably a trans-cyclooctenyl group(also referred to as a TCO group) and most preferably atrans-cyclooctenyl group according to formula (9zi) or (9zj) as shownbelow. In another preferred embodiment, the cycloalkenyl group is acyclopropenyl group, wherein the cyclopropenyl group is optionallysubstituted. In another preferred embodiment, the cycloalkenyl group isa norbornenyl group, an oxanorbomenyl group, a norbornadienyl group oran oxanorbornadienyl group, wherein the norbornenyl group, oxanorbomenylgroup, norbornadienyl group or an oxanorbornadienyl group is optionallysubstituted. In a further preferred embodiment, the cycloalkenyl groupis according to formula (9k), (9l), (9m) or (9zc) as shown below,wherein T is CH₂ or O, R⁹ is independently selected from the groupconsisting of hydrogen, a linear or branched C₁-C₁₂ alkyl group or aC₄-C₁₂ (hetero)aryl group, and R¹⁹ is selected from the group consistingof hydrogen and fluorinated hydrocarbons. Preferably, R⁹ isindependently hydrogen or a C₁-C₆ alkyl group, more preferably R⁹ isindependently hydrogen or a C₁-C₄ alkyl group. Even more preferably R⁹is independently hydrogen or methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl or t-butyl. Yet even more preferably R⁹ is independentlyhydrogen or methyl. In a further preferred embodiment, R¹⁹ is selectedfrom the group of hydrogen and —CF₃, —C₂F₅, —C₃F₇ and —C₄F₉, morepreferably hydrogen and —CF₃. In a further preferred embodiment, thecycloalkenyl group is according to formula (9k), wherein one R⁹ ishydrogen and the other R₉ is a methyl group. In another furtherpreferred embodiment, the cycloalkenyl group is according to formula(9l), wherein both R⁹ are hydrogen. In these embodiments it is furtherpreferred that 1 is 0 or 1. In another further preferred embodiment, thecycloalkenyl group is a norbornenyl (T is CH₂) or an oxanorbomenyl (T isO) group according to formula (9m), or a norbornadienyl (T is CH₂) or anoxanorbornadienyl (T is O) group according to formula (9zc), wherein R⁹is hydrogen and R¹⁹ is hydrogen or —CF₃, preferably —CF₃.

In another preferred embodiment, Q¹ is a (hetero)cycloalkynyl group. The(hetero)cycloalkynyl group is optionally substituted. Preferably, the(hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, i.e. aheterocyclooctynyl group or a cyclooctynyl group, wherein the(hetero)cyclooctynyl group is optionally substituted. In a furtherpreferred embodiment, the (hetero)cyclooctynyl group is according toformula (9n), also referred to as a DIBO group, (9o), also referred toas a DIBAC group or (9p), also referred to as a BARAC group, or (9zk),also referred to as a COMBO group, all as shown below, wherein U is O orNR⁹, and preferred embodiments of R⁹ are as defined above. The aromaticrings in (9n) are optionally O-sulfonylated at one or more positions,whereas the rings of (9o) and (9p) may be halogenated at one or morepositions.

In an especially preferred embodiment, the nitrogen atom attached to R¹in compound (4b) is the nitrogen atom in the ring of theheterocycloalkyne group such as the nitrogen atom in 9o. In other words,c, d and g are 0 in compound (4b) and R¹ and Q¹, together with thenitrogen atom they are attached to, form a heterocycloalkyne group,preferably a heterocyclooctyne group, most preferably theheterocyclooctyne group according to formula (9o) or (9p). Herein, thecarbonyl moiety of (9o) is replaced by the sulfonyl group of the groupaccording to formula (1). Alternatively, the nitrogen atom to which R¹is attached is the same atom as the atom designated as U in formula(9n). In other words, when Q¹ is according to formula (9n), U may be theright nitrogen atom of the group according to formula (1), or U=NR⁹ andR⁹ is the remainder of the group according to formula (1) and R¹ is thecyclooctyne moiety.

In another preferred embodiment, Q¹ is an, optionally substituted,bicyclo[6.1.0] non-4-yn-9-yl] group, also referred to as a BCN group.Preferably, the bicyclo[6.1.0] non-4-yn-9-yl] group is according toformula (9q) as shown below.

In another preferred embodiment, Q¹ is a conjugated (hetero)diene groupcapable of reacting in a Diels-Alder reaction. Preferred (hetero)dienegroups include optionally substituted tetrazinyl groups, optionallysubstituted 1,2-quinone groups and optionally substituted triazinegroups. More preferably, said tetrazinyl group is according to formula(9r), as shown below, wherein R⁹ is selected from the group consistingof hydrogen, a linear or branched C₁-C₁₂ alkyl group or a C₄-C₁₂(hetero)aryl group. Preferably, R⁹ is hydrogen, a C₁-C₆ alkyl group or aC₄-C₁₀ (hetero)aryl group, more preferably R⁹ is hydrogen, a C₁-C₄ alkylgroup or a C₄-C₆ (hetero)aryl group. Even more preferably R⁹ ishydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butylor pyridyl. Yet even more preferably R⁹ is hydrogen, methyl or pyridyl.More preferably, said 1,2-quinone group is according to formula (9zl) or(9zm). Said triazine group may be any regioisomer. More preferably, saidtriazine group is a 1,2,3-triazine group or a 1,2,4-triazine group,which may be attached via any possible location, such as indicated informula (9zn). The 1,2,3-triazine is most preferred as triazine group.In another preferred embodiment, Q¹ is an azido group according toformula (9s) as shown below.

In another preferred embodiment, Q¹ is an, optionally substituted,triarylphosphine group that is suitable to undergo a Staudinger ligationreaction. Preferably, the phosphine group is according to formula (9t)as shown below, wherein R¹⁰ is hydrogen or a (thio)ester group. When R¹⁰is a (thio)ester group, it is preferred that R¹⁰ is —C(O)—V—R¹¹, whereinV is O or S and R¹¹ is a C₁-C₁₂ alkyl group. Preferably, R¹¹ is a C₁-C₆alkyl group, more preferably a C₁-C₄ alkyl group. Most preferably, R¹¹is a methyl group.

In another preferred embodiment, Q¹ is a nitrile oxide group accordingto formula (9u) as shown below.

In another preferred embodiment, Q¹ is a nitrone group. Preferably, thenitrone group is according to formula (9v) as shown below, wherein R¹²is selected from the group consisting of linear or branched C₁-C₁₂ alkylgroups and C₆-C₁₂ aryl groups. Preferably, R¹² is a C₁-C₆ alkyl group,more preferably R¹² is a C₁-C₄ alkyl group. Even more preferably R¹² ismethyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Yet evenmore preferably R¹² is methyl.

In another preferred embodiment, Q¹ is a nitrile imine group.Preferably, the nitrile imine group is according to formula (9w) or(9zd) as shown below, wherein R¹³ is selected from the group consistingof linear or branched C₁-C₁₂ alkyl groups and C₆-C₁₂ aryl groups.Preferably, R¹³ is a C₁-C₆ alkyl group, more preferably R¹³ is a C₁-C₄alkyl group. Even more preferably R¹³ is methyl, ethyl, n-propyl,i-propyl, n-butyl, s-butyl or t-butyl. Yet even more preferably R¹³ ismethyl.

In another preferred embodiment, Q¹ is a diazo group. Preferably, thediazo group is according to formula (9x) as shown below, wherein R¹⁴ isselected from the group consisting of hydrogen or a carbonyl derivative.More preferably, R¹⁴ is hydrogen.

In another preferred embodiment, Q¹ is a ketone group. More preferably,the ketone group is according to formula (9y) as shown below, whereinR¹⁵ is selected from the group consisting of linear or branched C₁-C₁₂alkyl groups and C₆-C₁₂ aryl groups. Preferably, R¹⁵ is a C₁-C₆ alkylgroup, more preferably R¹⁵ is a C₁-C₄ alkyl group. Even more preferablyR¹⁵ is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl.Yet even more preferably R¹⁵ is methyl.

In another preferred embodiment, Q¹ is an (O-alkyl)hydroxylamino group.More preferably, the (O-alkyl)hydroxylamino group is according toformula (9z) as shown below.

In another preferred embodiment, Q¹ is a hydrazine group. Preferably,the hydrazine group is according to formula (9za) as shown below.

In another preferred embodiment, Q¹ is a halogenated N-maleimidyl groupor a sulfonylated N-maleimidyl group. When Q¹ is a halogenated orsulfonylated N-maleimidyl group, Q¹ is preferably according to formula(9ze) as shown below, wherein R⁶ is independently selected from thegroup consisting of hydrogen F, Cl, Br, I and —S(O)₂R₁₈, wherein R¹⁸ isselected from the group consisting of, optionally substituted, C₁-C₁₂alkyl groups and C₄-C₁₂ (hetero)aryl groups, and with the proviso thatat least one R⁶ is not hydrogen. When R⁶ is halogen (i.e. when R⁶ is F,Cl, Br or I), it is preferred that R⁶ is Br. When R⁶ is —S(O)₂R₁₈, it ispreferred that R₁₈ is a C₁-C₆ alkyl group or a C₄-C₆ (hetero)aryl group,preferably a phenyl group.

In another preferred embodiment, Q¹ is a carbonyl halide group accordingto formula (9zf) as shown below, wherein X is selected from the groupconsisting of F, Cl, Br and I. Preferably, X is Cl or Br, and mostpreferably, X is Cl.

In another preferred embodiment, Q¹ is an allenamide group according toformula (9zg).

In another preferred embodiment, Q¹ is a1,1-bis(sulfonylmethyl)methylcarbonyl group according to formula (9zh),or an elimination derivative thereof, wherein R¹⁸ is selected from thegroup consisting of, optionally substituted, C₁-C₁₂ alkyl groups andC₄-C₁₂ (hetero)aryl groups. More preferably, R₁₈ is an, optionallysubstituted, C₁-C₆ alkyl group or a C₄-C₆ (hetero)aryl group, and mostpreferably a phenyl group.

wherein k, l, X, T, U, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴,R¹⁵, R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are as defined above.

In a preferred embodiment of the conjugation process according to theinvention as described hereinbelow, conjugation is accomplished via acycloaddition, such as a Diels-Alder reaction or a 1,3-dipolarcycloaddition, preferably the 1,3-dipolar cycloaddition. According tothis embodiment, the reactive group Q¹ (as well as F¹ on thebiomolecule) is selected from groups reactive in a cycloadditionreaction. Herein, reactive groups Q¹ and F¹ are complementary, i.e. theyare capable of reacting with each other in a cycladdition reaction.

For a Diels-Alder reaction, one of F¹ and Q¹ is a diene and the other ofF¹ and Q¹ is a dienophile. As appreciated by the skilled person, theterm “diene” in the context of the Diels-Alder reaction refers to1,3-(hetero)dienes, and includes conjugated dienes (R₂C═CR—CR═CR₂),imines (e.g. R₂C═CR—N═CR₂ or R₂C═CR—CR═NR, R₂C═N—N═CR₂) and carbonyls(e.g. R₂C═CR—CR═O or O═CR—CR═O). Hetero-Diels-Alder reactions with N-and O-containing dienes are known in the art. Any diene known in the artto be suitable for Diels-Alder reactions may be used as reactive groupQ¹ or F¹. Preferred dienes include tetrazines as described above,1,2-quinones as described above and triazines as described above.Although any dienophile known in the art to be suitable for Diels-Alderreactions may be used as reactive groups Q¹ or F¹, the dienophile ispreferably an alkene or alkyne group as described above, most preferablyan alkyne group. For conjugation via a Diels-Alder reaction, it ispreferred that F¹ is the diene and Q¹ is the dienophile. Herein, when Q¹is a diene, F¹ is a dienophile and when Q¹ is a dienophile, F¹ is adiene. Most preferably, Q¹ is a dienophile, preferably Q¹ is orcomprises an alkynyl group, and F¹ is a diene, preferably a tetrazine,1,2-quinone or triazine group.

For a 1,3-dipolar cycloaddition, one of F¹ and Q¹ is a 1,3-dipole andthe other of and Q¹ is a dipolarophile. Any 1,3-dipole known in the artto be suitable for 1,3-dipolar cycloadditions may be used as reactivegroup Q¹ or F¹. Preferred 1,3-dipoles include azido groups, nitronegroups, nitrile oxide groups, nitrile imine groups and diazo groups.Although any dipolarophile known in the art to be suitable for1,3-dipolar cycloadditions may be used as reactive groups Q¹ or F¹, thedipolarophile is preferably an alkene or alkyne group, most preferablyan alkyne group. For conjugation via a 1,3-dipolar cycloaddition, it ispreferred that F¹ is the 1,3-dipole and Q¹ is the dipolarophile. Herein,when Q¹ is a 1,3-dipole, F¹ is a dipolarophile and when Q¹ is adipolarophile, F¹ is a 1,3-dipole. Most preferably, Q¹ is adipolarophile, preferably Q¹ is or comprises an alkynyl group, and F¹ isa 1,3-dipole, preferably an azido group. Thus, in a preferredembodiment, Q¹ is selected from dipolarophiles and dienophiles.Preferably, Q¹ is an alkene or an alkyne group. In an especiallypreferred embodiment, Q¹ comprises an alkyne group, preferably selectedfrom the alkynyl group as described above, the cycloalkenyl group asdescribed above, the (hetero)cycloalkynyl group as described above and abicyclo[6.1.0]non-4-yn-9-yl] group, more preferably Q¹ is selected fromthe formulae (9j), (9n), (9o), (9p), (9q) and (9zk), as defined aboveand depicted below, more preferably selected from the formulae (9n),(9o), (9p), (9q) and (9zk). Most preferably, Q¹ is abicyclo[6.1.0]non-4-yn-9-yl] group, preferably of formula (9q). Thesegroups are known to be highly effective in the conjugation withazido-functionalized biomolecules as described herein, and when thesulfamide linker according to the invention is employed in suchlinker-conjugates and bioconjugates, any aggregation is beneficiallyreduced to a minimum. The sulfamide linker according to the inventionprovides a significant reduction in aggregation especially for suchhydrophobic reactive groups of Q¹, and for the conjugated bioconjugates.

As was described above, in the compound according to the invention, Q¹is capable of reacting with a reactive group F¹ that is present on abiomolecule. Complementary reactive groups F¹ for reactive group Q¹ areknown to a person skilled in the art, and are described in more detailbelow. Some representative examples of reaction between F¹ and Q¹ andtheir corresponding products are depicted in FIG. 22.

As described above, target molecule D and reactive group Q¹ arecovalently attached to the linker in the linker-conjugate according tothe invention. Covalent attachment of a target molecule D to the linkermay occur for example via reaction of a functional group F² present onthe target molecule with a reactive group Q² present on the linker.Suitable organic reactions for the attachment of a target molecule D toa linker are known to a person skilled in the art, as are functionalgroups F² that are complementary to a reactive group Q². Consequently, Dmay be attached to the linker via a connecting group Z.

The term “connecting group” herein refers to the structural elementconnecting one part of a compound and another part of the same compound.As will be understood by the person skilled in the art, the nature of aconnecting group depends on the type of organic reaction with which theconnection between the parts of said compound was obtained. As anexample, when the carboxyl group of R—C(O)—OH is reacted with the aminogroup of H₂N—R′ to form R—C(O)—N(H)—R′, R is connected to R′ viaconnecting group Z, and Z may be represented by the group —C(O)—N(H)—.

Reactive group Q¹ may be attached to the linker in a similar manner.Consequently, Q¹ may be attached to the spacer-moiety via a connectinggroup Z.

Numerous reactions are known in the art for the attachment of a targetmolecule to a linker, and for the attachment of a reactive group Q¹ to alinker. Consequently, a wide variety of connecting groups Z may bepresent in the linker-conjugate according to the invention.

The invention thus also relates to a compound according to the formula:(Q¹)_(y)-(Z^(w))—Sp-(Z^(x))-(D)_(z),wherein:y is an integer in the range of 1 to 10;z is an integer in the range of 1 to 10;Q¹ is a reactive group capable of reacting with a functional group F¹present on a biomolecule;D is a target molecule;Sp is a spacer moiety, wherein a spacer moiety is defined as a moietythat spaces (i.e. provides a certain distance between) and covalentlylinks reactive group Q¹ and target molecule D;Z^(w) is a connecting group connecting reactive group Q¹ to said spacermoiety;Z^(x) is a connecting group connecting target molecule D to said spacermoiety; andwherein said spacer moiety comprises a group according to Formula (1) ora salt thereof, wherein the group according to Formula (1) is as definedabove.

In a preferred embodiment, a in the group according to formula (1) is 0.In another preferred embodiment, a in the group according to formula (1)is 1.

Preferred embodiments for y and z are as defined above for(Q¹)_(y)-Sp-(D)_(z). It is further preferred that the compound isaccording to the formula Q¹-(Z^(w))—Sp-(Z^(x))-(D)₄,Q¹-(Z^(w))-Sp-(Z^(x))-(D)₃, Q¹-(Z^(w))-Sp-(Z^(x))-(D)₂ orQ¹-(Z^(w))-Sp-(Z^(x))-D, more preferably Q¹-(Z^(w))-Sp-(Z^(x))-(D)₂ orQ¹-(Z^(w))-Sp-(Z^(x))-D and most preferably Q¹-(Z^(w))-Sp-(Z^(x))-D,wherein Z^(w) and Z^(x) are as defined above.

Preferably, Z^(w) and Z^(x) are independently selected from the groupconsisting of —O—, —S—, —NR²—, —N═N—, —C(O)—, —C(O)—NR²—, —O—C(O)—,—O—C(O)—O—, —O—C(O)—NR², —NR₂—C(O)—, —NR²—C(O)—O—, —NR²—C(O)—NR²—,—S—C(O)—, —S—C(O)—O—, —S—C(O)—NR²—, —S(O)—, —S(O)₂—, —O—S(O)₂—,—O—S(O)₂—O—, —O—S(O)₂—NR²—, —O—S(O)—, —O—S(O)—O—, —O—S(O)—NR²—,—O—NR²—C(O)—, —O—NR²—C(O)—O—, —O—NR²—C(O)—NR²—, —NR²—O—C(O)—,—NR²—O—C(O)—O—, —NR²—O—C(O)—NR²—, —O—NR²—C(S)—, —O—NR²—C(S)—O—,—O—NR²—C(S)—NR²—, —NR²—O—C(S)—, —NR²—O—C(S)—O—, —NR²—O—C(S)—NR²—,—O—C(S)—, —O—C(S)—O—, —O—C(S)—NR²—, —NR²—C(S)—,—NR²—C(S)—O—,—NR²—C(S)—NR²—, —S—S(O)₂—, —S—S(O)₂—O—, —S—S(O)₂—NR²—,—NR²—O—S(O)—, —NR²—O—S(O)—O—, —NR²—O—S(O)—NR²—, —NR²—O—S(O)₂—,—NR²—O—S(O)₂—O—, —NR²—O—S(O)₂—NR²—, —O—NR²—S(O)—, —O—NR²—S(O)—O—,—O—NR²—S(O)—NR²—, —O—NR²—S(O)₂—O—, —O—NR²—S(O)₂—NR²—, —O—NR²—S(O)₂—,—O—P(O)(R²)₂—, —S—P(O)(R²)₂—, —NR²—P(O)(R²)₂— and combinations of two ormore thereof, wherein R² is independently selected from the groupconsisting of hydrogen, C₁-C₂₄ alkyl groups, C₂-C₂₄ alkenyl groups,C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkyl groups, the alkyl groups,alkenyl groups, alkynyl groups and cycloalkyl groups being optionallysubstituted.

Preferred embodiments for D and Q¹ are as defined above.

More particularly, the present invention relates to a compound accordingto formula (4a) or (4b), or a salt thereof:

wherein:

-   -   a is independently 0 or 1;    -   b is independently 0 or 1;    -   c is 0 or 1;    -   d is 0 or 1;    -   e is 0 or 1;    -   f is an integer in the range of 1 to 150;    -   g is 0 or 1;    -   i is 0 or 1;    -   D is a target molecule;    -   Q¹ is a reactive group capable of reacting with a functional        group F¹ present on a biomolecule;    -   Sp¹ is a spacer moiety;    -   Sp² is a spacer moiety;    -   Sp³ is a spacer moiety;    -   Sp⁴ is a spacer moiety;    -   Z¹ is a connecting group that connects Q¹ or Sp³ to Sp², O or        C(O) or N(R¹);    -   Z² is a connecting group that connects D or Sp⁴ to Sp¹, N(R¹), 0        or C(O); and    -   R¹ is selected from the group consisting of hydrogen, C₁-C₂₄        alkyl groups, C₃-C₂₄ cycloalkyl groups, C₁-C₂₄ (hetero)aryl        groups, C₁-C₂₄ alkyl(hetero)aryl groups and C₁-C₂₄        (hetero)arylalkyl groups, the C₁-C₂₄ alkyl groups, C₃-C₂₄        cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups; or R¹ is D,        -[(Sp¹)_(b)-(Z²)^(e)-(Sp⁴)^(i)-D] or        -[(Sp²)_(c)-(Z¹)_(d)-(Sp³)_(g)-Q¹], wherein Sp¹, Sp², Sp³, Sp⁴,        Z¹, Z², D, Q¹, b, c, d, e, g and i are as defined above.

The compound according to formula (4a) or (4b), or a salt thereof, mayalso be referred to as a linker-conjugate.

In a preferred embodiment, a is 1 in the compound according to formula(4a) or (4b). In another preferred embodiment, a is 0 in the compoundaccording to formula (4a) or (4b).

As defined above, Z¹ is a connecting group that connects Q¹ or Sp³ toSp², O or C(O) or N(R¹), and Z² is a connecting group that connects D orSp⁴ to Sp¹, N(R¹), 0 or C(O). As described in more detail above, theterm “connecting group” refers to a structural element connecting onepart of a compound and another part of the same compound.

In a compound according to formula (4a), connecting group Z¹, whenpresent (i.e. when d is 1), connects Q¹ (optionally via a spacer moietySp³) to the O-atom or the C(O) group of the compound according toformula (4a), optionally via a spacer moiety Sp². More particularly,when Z¹ is present (i.e. d is 1), and when Sp³ and Sp² are absent (i.e.g is 0 and c is 0), Z¹ connects Q¹ to the O-atom (a is 1) or to the C(O)group (a is 0) of the linker-conjugate according to formula (4a). WhenZ¹ is present (i.e. when d is 1), Sp³ is present (i.e. g is 1) and Sp²is absent (i.e. c is 0), Z¹ connects spacer moiety Sp³ to the O-atom (ais 1) or to the C(O) group (a is 0) of the linker-conjugate according toformula (4a). When Z¹ is present (i.e. d is 1), and when Sp³ and Sp² arepresent (i.e. g is 1 and c is 1), Z¹ connects spacer moiety Sp³ tospacer moiety Sp² of the linker-conjugate according to formula (4a).When Z¹ is present (i.e. when d is 1), Sp³ is absent (i.e. g is 0) andSp² is present (i.e. c is 1), Z¹ connects Q¹ to spacer moiety Sp² of thelinker-conjugate according to formula (4a).

In a compound according to formula (4b), connecting group Z¹, whenpresent (i.e. when d is 1), connects Q¹ (optionally via a spacer moietySp³) to the N-atom of the N(R¹) group in the linker-conjugate accordingto formula (4b), optionally via a spacer moiety Sp². More particularly,when Z¹ is present (i.e. d is 1), and when Sp³ and Sp² are absent (i.e.g is 0 and c is 0), Z¹ connects Q¹ to the N-atom of the N(R¹) group ofthe linker-conjugate according to formula (4b). When Z¹ is present (i.e.when d is 1), Sp³ is present (i.e. g is 1) and Sp² is absent (i.e. c is0), Z¹ connects spacer moiety Sp³ to the N-atom of the N(R¹) group ofthe linker-conjugate according to formula (4b). When Z¹ is present (i.e.d is 1), and when Sp³ and Sp² are present (i.e. g is 1 and c is 1), Z¹connects spacer moiety Sp³ to spacer moiety Sp² of the linker-conjugateaccording to formula (4b). When Z¹ is present (i.e. when d is 1), Sp³ isabsent (i.e. g is 0) and Sp² is present (i.e. c is 1), Z¹ connects Q¹ tospacer moiety Sp² of the linker-conjugate according to formula (4b).

In the compound according to formula (4a), when c, d and g are all 0,then Q¹ is attached directly to the O-atom (when a is 1) or to the C(O)group (when a is 0) of the linker-conjugate according to formula (4a).

In the compound according to formula (4b), when c, d and g are all 0,then Q¹ is attached directly to the N-atom of the N(R¹) group of thelinker-conjugate according to formula (4b).

In a compound according to formula (4a), connecting group Z², whenpresent (i.e. when e is 1), connects D (optionally via a spacer moietySp⁴) to the N-atom of the N(R¹) group in the linker-conjugate accordingto formula (4a), optionally via a spacer moiety Sp′. More particularly,when Z² is present (i.e. e is 1), and when Sp′ and Sp⁴ are absent (i.e.b is 0 and i is 0), Z² connects D to the N-atom of the N(R¹) group ofthe linker-conjugate according to formula (4a). When Z² is present (i.e.when e is 1), Sp⁴ is present (i.e. i is 1) and Sp′ is absent (i.e. b is0), Z² connects spacer moiety Sp⁴ to the N-atom of the N(R¹) group ofthe linker-conjugate according to formula (4a). When Z² is present (i.e.e is 1), and when Sp′ and Sp⁴ are present (i.e. b is 1 and i is 1), Z²connects spacer moiety Sp′ to spacer moiety Sp⁴ of the linker-conjugateaccording to formula (4a). When Z² is present (i.e. when e is 1), Sp⁴ isabsent (i.e. i is 0) and Sp′ is present (i.e. b is 1), Z² connects D tospacer moiety Sp′ of the linker-conjugate according to formula (4a).

In the compound according to formula (4a), when b, e and i are all 0,then D is attached directly to N-atom of the N(R¹) group of thelinker-conjugate according to formula (4a).

In the compound according to formula (4b), when b, e and i are all 0,then D is attached directly to the O-atom (when a is 1) or to the C(O)group (when a is 0) of the linker-conjugate according to formula (4b).

As will be understood by the person skilled in the art, the nature of aconnecting group depends on the type of organic reaction with which theconnection between the specific parts of said compound was obtained. Alarge number of organic reactions are available for connecting areactive group Q¹ to a spacer moiety, and for connecting a targetmolecule to a spacer-moiety. Consequently, there is a large variety ofconnecting groups Z¹ and Z².

In a preferred embodiment of the linker-conjugate according to formula(4a) and (4b), Z¹ and Z² are independently selected from the groupconsisting of —O—, —S—, —S—S—, —NR²—, —N═N—, —C(O)—, —C(O)—NR²—,—O—C(O)—, —O—C(O)—O—, —O—C(O)—NR², —NR₂—C(O)—, —NR²—C(O)—O—,—NR²—C(O)—NR²—, —S—C(O)—, —S—C(O)—O—, —S—C(O)—NR²—, —S(O)—, —S(O)₂—,—O—S(O)₂—, —O—S(O)₂—O—, —O—S(O)₂—NR²—, —O—S(O)—, —O—S(O)—O—,—O—S(O)—NR²—, —O—NR²—C(O)—, —O—NR²—C(O)—O—, —O—NR²—C(O)—NR²—,—NR²—O—C(O)—, —NR²—O—C(O)—O—, —NR²—O—C(O)—NR²—, —O—NR²—C(S)—,—O—NR²—C(S)—O—, —O—NR²—C(S)—NR²—, —NR²—O—C(S)—, —NR²—O—C(S)—O—,—NR²—O—C(S)—NR²—, —O—C(S)—, —O—C(S)—O—, —O—C(S)—NR²—, —NR²—C(S)—,—NR²—C(S)—O—, —NR²—C(S)—NR²—, —S—S(O)₂—, —S—S(O)₂—O—, —S—S(O)₂—NR²—,—NR²—O—S(O)—, —NR²—O—S(O)—O—, —NR²—O—S(O)—NR²—, —NR²—O—S(O)₂—,—NR²—O—S(O)₂—O—, —NR²—O—S(O)₂—NR²—, —O—NR²—S(O)—, —O—NR²—S(O)—O—,—O—NR²—S(O)—NR²—, —O—NR²—S(O)₂—O—, —O—NR²—S(O)₂—NR²—, —O—NR²—S(O)₂—,—O—P(O)(R²)₂—, —S—P(O)(R²)₂—, —NR²—P(O)(R²)₂— and combinations of two ormore thereof, wherein R² is independently selected from the groupconsisting of hydrogen, C₁-C₂₄ alkyl groups, C₂-C₂₄ alkenyl groups,C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkyl groups, the alkyl groups,alkenyl groups, alkynyl groups and cycloalkyl groups being optionallysubstituted.

As described above, in the compound according to formula (4a) or (4b),Sp¹, Sp², Sp³ and Sp⁴ are spacer-moieties. Sp¹, Sp², Sp³ and Sp⁴ may be,independently, absent or present (b, c, g and i are, independently, 0 or1). Sp¹, if present, may be different from Sp², if present, from Sp³and/or from Sp⁴, if present.

Spacer-moieties are known to a person skilled in the art. Examples ofsuitable spacer-moieties include (poly)ethylene glycol diamines (e.g.1,8-diamino-3,6-dioxaoctane or equivalents comprising longer ethyleneglycol chains), polyethylene glycol chains or polyethylene oxide chains,polypropylene glycol chains or polypropylene oxide chains and1,x-diaminoalkanes wherein x is the number of carbon atoms in thealkane.

Another class of suitable spacer-moieties comprises cleavablespacer-moieties, or cleavable linkers. Cleavable linkers are well knownin the art. For example Shabat et al., Soft Matter 2012, 6, 1073,incorporated by reference herein, discloses cleavable linkers comprisingself-immolative moieties that are released upon a biological trigger,e.g. an enzymatic cleavage or an oxidation event. Some examples ofsuitable cleavable linkers are disulfide-linkers that are cleaved uponreduction, peptide-linkers that are cleaved upon specific recognition bya protease, e.g. cathepsin, plasmin or metalloproteases, orglycoside-based linkers that are cleaved upon specific recognition by aglycosidase, e.g. glucoronidase, or nitroaromatics that are reduced inoxygen-poor, hypoxic areas. Herein, suitable cleavable spacer-moietiesalso include spacer moieties comprising a specific, cleavable, sequenceof amino acids. Examples include e.g. spacer-moieties comprising aVal-Cit (valine-citrulline) moiety. Bioconjugates containing a cleavablelinker, such as Val-Cit linker, in particular Val-Cit-PABC, sufferconsiderably from aggregation in view of their limited water-solubility.For such bioconjugates, incorporating the sulfamide linker according tothe invention is particularly beneficial. Also, conjugation reactionswith a linker-conjugate comprising a cleavable linker are hampered bythe limited water-solubility of the linker-conjugate. Hence,linker-conjugates comprising a cleavable linker, such as Val-Cit linker,in particular Val-Cit-PABC, and the sulfamide linker according to theinvention outperform linker-conjugates comprising such a cleavablelinker but lacking such sulfamide linker in conjugation to biomolecules.

Thus, in a preferred embodiment of the linker-conjugates according toformula (4a) and (4b), spacer moieties Sp¹, Sp², Sp³ and/or Sp⁴, ifpresent, comprise a sequence of amino acids. Spacer-moieties comprisinga sequence of amino acids are known in the art, and may also be referredto as peptide linkers. Examples include spacer-moieties comprising aVal-Cit moiety, e.g. Val-Cit-PABC, Val-Cit-PAR, Fmoc-Val-Cit-PAB, etc.Preferably, a Val-Cit-PABC moiety is employed in the linker-conjugate.

In a preferred embodiment of the linker-conjugates according to formula(4a) and (4b), spacer moieties Sp¹, Sp², Sp³ and Sp⁴, if present, areindependently selected from the group consisting of linear or branchedC₁-C₂₀₀ alkylene groups, C₂-C₂₀₀ alkenylene groups, C₂-C₂₀₀ alkynylenegroups, C₃-C₂₀₀ cycloalkylene groups, C₅-C₂₀₀ cycloalkenylene groups,C₈-C₂₀₀ cycloalkynylene groups, C₇-C₂₀₀ alkylarylene groups, C₇-C₂₀₀arylalkylene groups, C₈-C₂₀₀ arylalkenylene groups and C₉-C₂₀₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted. When the alkylene groups,alkenylene groups, alkynylene groups, cycloalkylene groups,cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups,arylalkylene groups, arylalkenylene groups and arylalkynylene groups areinterrupted by one or more heteroatoms as defined above, it is preferredthat said groups are interrupted by one or more O-atoms, and/or by oneor more S—S groups.

More preferably, spacer moieties Sp¹, Sp², Sp³ and Sp⁴, if present, areindependently selected from the group consisting of linear or branchedC₁-C₁₀₀ alkylene groups, C₂-C₁₀₀ alkenylene groups, C₂-C₁₀₀ alkynylenegroups, C₃-C₁₀₀ cycloalkylene groups, C₅-C₁₀₀ cycloalkenylene groups,C₈-C₁₀₀ cycloalkynylene groups, C₇-C₁₀₀ alkylarylene groups, C₇-C₁₀₀arylalkylene groups, C₈-C₁₀₀ arylalkenylene groups and C₉-C₁₀₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

Even more preferably, spacer moieties Sp¹, Sp², Sp³ and Sp⁴, if present,are independently selected from the group consisting of linear orbranched C₁-C₅₀ alkylene groups, C₂-C₅₀ alkenylene groups, C₂-C₅alkynylene groups, C₃-C₅₀ cycloalkylene groups, C₅-C₅₀ cycloalkenylenegroups, C₈-C₅₀ cycloalkynylene groups, C₇-C₅₀ alkylarylene groups,C₇-C₅₀ arylalkylene groups, C₈-C₅₀ arylalkenylene groups and C₉-C₅₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

Yet even more preferably, spacer moieties Sp¹, Sp², Sp^(a) and Sp⁴, ifpresent, are independently selected from the group consisting of linearor branched C₁-C₂₀ alkylene groups, C₂-C₂₀ alkenylene groups, C₂-C₂₀alkynylene groups, C₃-C₂₀ cycloalkylene groups, C₅-C₂₀ cycloalkenylenegroups, C₈-C₂₀ cycloalkynylene groups, C₇-C₂₀ alkylarylene groups,C₇-C₂₀ arylalkylene groups, C₈-C₂₀ arylalkenylene groups and C₉-C₂₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

In these preferred embodiments it is further preferred that the alkylenegroups, alkenylene groups, alkynylene groups, cycloalkylene groups,cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups,arylalkylene groups, arylalkenylene groups and arylalkynylene groups areunsubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, preferably O, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups, preferably hydrogen or methyl.

Most preferably, spacer moieties Sp¹, Sp², Sp³ and Sp⁴, if present, areindependently selected from the group consisting of linear or branchedC₁-C₂₀ alkylene groups, the alkylene groups being optionally substitutedand optionally interrupted by one or more heteroatoms selected from thegroup of O, S and NR³, wherein R³ is independently selected from thegroup consisting of hydrogen, C₁-C₂₄ alkyl groups, C₂-C₂₄ alkenylgroups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkyl groups, the alkylgroups, alkenyl groups, alkynyl groups and cycloalkyl groups beingoptionally substituted. In this embodiment, it is further preferred thatthe alkylene groups are unsubstituted and optionally interrupted by oneor more heteroatoms selected from the group of O, S and NR³, preferablyO and/or or S—S, wherein R³ is independently selected from the groupconsisting of hydrogen and C₁-C₄ alkyl groups, preferably hydrogen ormethyl.

Preferred spacer moieties Sp¹, Sp², Sp³ and Sp⁴ thus include —(CH₂)_(n),—(CH₂CH₂)_(n)—, —(CH₂CH₂O)_(n)—, —(OCH₂CH₂)_(n)—, —(CH₂CH₂O)_(n)CH₂CH₂—,—CH₂CH₂(OCH₂CH₂)_(n)—, —(CH₂CH₂CH₂O)_(n)—, —(OCH₂CH₂CH₂)_(n)—,—(CH₂CH₂CH₂O)_(n)CH₂CH₂CH₂— and —CH₂CH₂CH₂(OCH₂CH₂CH₂)_(n)—, wherein nis an integer in the range of 1 to 50, preferably in the range of 1 to40, more preferably in the range of 1 to 30, even more preferably in therange of 1 to 20 and yet even more preferably in the range of 1 to 15.More preferably n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1,2, 3, 4, 5, 6, 7 or 8, even more preferably 1, 2, 3, 4, 5 or 6, yet evenmore preferably 1, 2, 3 or 4.

Since Sp¹, Sp², Sp^(a) and Sp⁴ are independently selected, Sp¹, ifpresent, may be different from Sp², if present, from Sp^(a) and/or fromSp⁴, if present.

Reactive groups Q¹ are described in more detail above. In thelinker-conjugate according to formula (4a) and (4b), it is preferredthat reactive group Q¹ is selected from the group consisting of,optionally substituted, N-maleimidyl groups, halogenated N-alkylamidogroups, sulfonyloxy N-alkylamido groups, ester groups, carbonate groups,sulfonyl halide groups, thiol groups or derivatives thereof, alkenylgroups, alkynyl groups, (hetero)cycloalkynyl groups,bicyclo[6.1.0]non-4-yn-9-yl] groups, cycloalkenyl groups, tetrazinylgroups, azido groups, phosphine groups, nitrile oxide groups, nitronegroups, nitrile imine groups, diazo groups, ketone groups,(O-alkyl)hydroxylamino groups, hydrazine groups, halogenatedN-maleimidyl groups, carbonyl halide groups, allenamide groups and1,1-bis(sulfonylmethyl)methylcarbonyl groups or elimination derivativesthereof. In a further preferred embodiment, Q¹ is according to formula(9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l),(9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x),(9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh),(9zi), (9zj), (9zk), (9zl), (9zm) or (9zn), wherein (9a), (9b), (9c),(9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o),(9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za),(9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk),(9zl), (9zm), (9zn) and preferred embodiments thereof, are as definedabove. In a preferred embodiment, Q¹ is according to formula (9a), (9b),(9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9zh), (9r),(9zl), (9zm) or (9zn). In an even further preferred embodiment, Q¹ isaccording to formula (9a), (9n), (9o), (9q), (9p), (9t), (9zh) or (9s),and in a particularly preferred embodiment, Q¹ is according to formula(9a), (9q), (9n), (9o), (9p), (9t) or (9zh), and preferred embodimentsthereof, as defined above.

Target molecule D and preferred embodiments for target molecule D in thelinker-conjugate according to formula (4a) and (4b) are as definedabove. In a preferred embodiment of the linker-conjugates according toformula (4a) and (4b), D is selected from the group consisting of anactive substance, a reporter molecule, a polymer, a solid surface, ahydrogel, a nanoparticle, a microparticle and a biomolecule. Activesubstances, reporter molecules, polymers, hydrogels, solid surfaces,nano- and microparticles and biomolecules, and preferred embodimentsthereof, are described in more detail above.

As described above, R¹ is selected from the group consisting ofhydrogen, C₁-C₂₄ alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄(hetero)aryl groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄(hetero)arylalkyl groups, the C₁-C₂₄ alkyl groups, C₃-C₂₄ cycloalkylgroups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄ alkyl(hetero)aryl groups andC₃-C₂₄ (hetero)arylalkyl groups optionally substituted and optionallyinterrupted by one or more heteroatoms selected from O, S and NR³wherein R³ is independently selected from the group consisting ofhydrogen and C₁-C₄ alkyl groups, or R¹ is D,-[(Sp¹)_(b)-(Z²)_(e)-(Sp⁴)_(i)-D] or -[(Sp²)_(c)-(Z¹)_(d)-(Sp³)_(g)-Q¹],wherein Sp¹, Sp², Sp^(a), Sp⁴, Z¹, Z², D, Q¹, b, c, d, e, g and i are asdefined above.

In a preferred embodiment, R¹ is hydrogen or a C₁-C₂₀ alkyl group, morepreferably R¹ is hydrogen or a C₁-C₁₆ alkyl group, even more preferablyR¹ is hydrogen or a C₁-C₁₀ alkyl group, wherein the alkyl group isoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³, preferably O, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups. In a further preferred embodiment, R¹ is hydrogen. Inanother further preferred embodiment, R¹ is a C₁-C₂₀ alkyl group, morepreferably a C₁-C₁₆ alkyl group, even more preferably a C₁-C₁₀ alkylgroup, wherein the alkyl group is optionally interrupted by one or moreO-atoms, and wherein the alkyl group is optionally substituted with an—OH group, preferably a terminal —OH group. In this embodiment it isfurther preferred that R¹ is a polyethyleneglycol chain comprising aterminal —OH group. In another further preferred embodiment, R¹ is aC₁-C₁₂ alkyl group, more preferably a C₁-C₆ alkyl group, even morepreferably a C₁-C₄ alkyl group, and yet even more preferably R¹ isselected from the group consisting of methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl and t-butyl.

In another preferred embodiment, R¹ is a target molecule D,-[(Sp¹)_(b)-(Z²)_(e)(Sp⁴)_(i)-D] or -[(Sp²)_(c)-(Z¹)_(d)—(Sp³)_(g)-Q¹],wherein Sp¹, Sp², Sp³, Sp⁴, Z¹, Z², D, Q¹, b, c, d, e, g and i are asdefined above. When R¹ is D or -[(Sp¹)_(b)-(Z²)_(e)(Sp⁴)_(i)-D], it isfurther preferred that the linker-conjugate is according to formula(4a). In this embodiment, linker-conjugate (4a) comprises two targetmolecules D, which may be the same or different. When R¹ is-[(Sp¹)_(b)-(Z²)_(e)-(Sp⁴)_(i)-D], Sp¹, b, Z², e, Sp⁴, i and D in-[(Sp¹)_(b)-(Z²)_(e)-(Sp⁴)_(i)-D] may be the same or different from Sp¹,b, Z², e, Sp⁴, i and D in the other -[(Sp¹)_(b)-(Z²)_(e)-(Sp⁴)_(i)-D]that is attached to the N-atom of N(R¹). In a preferred embodiment, both-[(Sp¹)_(b)-(Z²)_(e)-(Sp⁴)_(i)-D] groups on the N-atom of N(R¹) are thesame.

When R¹ is -[(Sp²)_(c)-(Z¹)_(d)-(Sp³)_(g)-Q¹], it is further preferredthat the linker-conjugate is according to formula (4b). In thisembodiment, linker-conjugate (4b) comprises two target molecules Q¹,which may be the same or different. When R¹ is-[(Sp²)_(c)-(Z¹)_(d)-(Sp³)_(g)-Q¹], Sp², c, Z¹, d, Sp³, g and D in-[(Sp¹)_(b)-(Z²)_(e)-(Sp⁴)_(i)-D] may be the same or different from Sp¹,b, Z², e, Sp⁴, i and Q¹ in the other -[(Sp²)_(c)-(Z¹)_(d)-(Sp³)_(g)-Q¹]that is attached to the N-atom of N(R¹). In a preferred embodiment,-[(Sp²)_(c)-(Z¹)_(d)-(Sp³)_(g)-Q¹] groups on the N-atom of N(R¹) are thesame.

In the linker-conjugates according to formula (4a) and (4b), f is aninteger in the range of 1 to 150. The linker-conjugate may thus comprisemore than one group according to formula (1), the group according toformula (1) being as defined above. When more than one group accordingto formula (1) is present, i.e. When f is 2 or more, then a, b, Sp¹ andR¹ are independently selected. In other words, when f is 2 or more, eacha is independently 0 or 1, each b is independently 0 or 1, each Sp¹ maybe the same or different and each R¹ may be the same or different. In apreferred embodiment, f is an integer in the range of 1 to 100,preferably in the range of 1 to 50, more preferably in the range of 1 to25, and even more preferably in the range of 1 to 15. More preferably, fis 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, even more preferably f is 1, 2, 3,4, 5, 6, 7 or 8, yet even more preferably f is 1, 2, 3, 4, 5 or 6, yeteven more preferably f is 1, 2, 3 or 4, and most preferably f is 1 inthis embodiment. In another preferred embodiment, f is an integer in therange of 2 to 150, preferably in the range of 2 to 100, more preferablyin the range of 2 to 50, more preferably in the range of 2 to 25, andeven more preferably in the range of 2 to 15. More preferably, f is 2,3, 4, 5, 6, 7, 8, 9 or 10, even more preferably f is 2, 3, 4, 5, 6, 7 or8, yet even more preferably f is 2, 3, 4, 5 or 6, yet even morepreferably f is 2, 3 or 4, and most preferably f is 2 in thisembodiment.

As described above, in a preferred embodiment, a is 0 in the compoundaccording to formula (4a) or (4b). The invention therefore also relatesto a compound according to formula (6a) or (6b), or a salt thereof:

wherein a, b, c, d, e, f, g, i, D, Q¹, Sp¹, Sp², Sp³, Sp⁴, Z¹, Z² andR¹, and their preferred embodiments, are as defined above for (4a) and(4b).

As described above, in another preferred embodiment, a is 1 in thecompound according to formula (4a) or (4b). The invention therefore alsorelates to a compound according to formula (7a) or (7b), or a saltthereof:

wherein a, b, c, d, e, f, g, i, D, Q¹, Sp¹, Sp², Sp³, Sp⁴, Z¹, Z² andR¹, and their preferred embodiments, are as defined above for (4a) and(4b).

When Sp⁴ is absent in the linker-conjugate according to formula (4a),i.e. when i is 0, target molecule D is linked to Z² (when e is 1), toSp¹ (when e is 0 and b is 1) or to N(R¹) (when e is 0 and b is 0). WhenSp⁴ is absent in the linker-conjugate according to formula (4b), i.e.when i is 0, target molecule D is linked to Z² (when e is 1), to Sp¹(when e is 0 and b is 1), to the O-atom (when a is 1 and b and e are 0)or to the C(O) group (when a is 0 and b and e are 0). The inventiontherefore also relates to a linker-conjugate according to formula (4c)or (4d), or a salt thereof:

wherein:a, b, c, d, e, f, g, D, Q¹, Sp¹, Sp², Sp³, Z¹, Z² and R¹, and theirpreferred embodiments, are as defined above for (4a) and (4b).

In a preferred embodiment, in the linker-conjugate according to formula(4c) or (4d), a is 0. In another preferred embodiment, in thelinker-conjugate according to formula (4c) or (4d), a is 1.

In a specific embodiment of the linker-conjugate according to theinvention, particularly a linker-conjugate according to formula (4a),(4b), (4c), (4d), (6a), (6b), (7a) or (7b), Sp¹, Sp²Sp³ and Sp⁴, ifpresent, are independently selected from the group consisting of linearor branched C₁-C₂₀ alkylene groups, the alkylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group consisting of O, S and NR³, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups, and Q¹ is according to formula (9a), (9b), (9c), (9d),(9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p),(9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za),(9zb), (9zc), (9zd), (9ze), (9zf), (9zg) (9zh), (9zi), (9zj), (9zk),(9zl), (9zm) or (9zn), wherein (9a), (9b), (9c), (9d), (9e), (9f), (9g),(9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s),(9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd),(9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm), (9zn) andpreferred embodiments thereof, are as defined above. In a preferredembodiment, Q¹ is according to formula (9a), (9b), (9c), (9f), (9j),(9n), (9o), (9p), (9q), (9s) (9t), (9zh), (9r), (9zl), (9zm) or (9zn).In an even further preferred embodiment, Q¹ is according to formula(9a), (9n), (9o), (9p), (9q), (9t), (9zh) or (9s), and in a particularlypreferred embodiment, Q¹ is according to formula (9a), (9q), (9n), (9p),(9t), (9zh) or (9o), and preferred embodiments thereof, as definedabove.

A linker is herein defined as a moiety that connects two or moreelements of a compound.

Consequently, in the linker-conjugate according to formula (4a), (4b),(4c), (4d), (6a), (6b), (7a) or (7b) as defined above, linker as definedabove may be represented by formula (8a) and (8b), respectively:

As will be understood by the person skilled in the art, preferredembodiments of spacer-moieties (8a) and (8b) may depend on e.g. thenature of reactive groups Q¹ and D in the linker-conjugate, thesynthetic method to prepare the linker-conjugate (e.g. the nature ofcomplementary functional group F² on a target molecule), the nature of abioconjugate that is prepared using the linker-conjugate (e.g. thenature of complementary functional group F¹ on the biomolecule).

When Q¹ is for example a cyclooctynyl group according to formula (9n),(9o), (9p), (9q) or (9zk) as defined above, then preferably Sp³ ispresent (g is 1).

When for example the linker-conjugate was prepared via reaction of areactive group Q² that is a cyclooctynyl group according to formula(9n), (9o), (9p), (9q) or (9zk) with an azido functional group F², thenpreferably Sp⁴ is present (i is 1).

Furthermore, it is preferred that at least one of Sp¹, Sp², Sp³ and Sp⁴is present, i.e. at least one of b, c, g, and i is not 0. In anotherpreferred embodiment, at least one of Sp¹ and Sp⁴ and at least one ofSp² and Sp³ are present.

When f is 2 or more, it is preferred that Sp¹ is present (b is 1).

These preferred embodiments of the linker-moiety (8a) and (8b) also holdfor the linker-moiety in bioconjugates according to the invention asdescribed in more detail below.

Preferred embodiments of Sp¹, Sp², Sp³ and Sp⁴ are as defined above.

Linker-Construct

A linker-construct is herein defined as a compound wherein a reactivegroup Q¹ is covalently connected to a reactive group Q² via a linker. Alinker-construct comprises a reactive group Q¹ capable of reacting witha reactive group F¹ present on a biomolecule, and a reactive group Q²capable of reacting with a reactive group F² present on a targetmolecule. Q¹ and Q² may be the same, or different. A linker-constructmay comprise more than one reactive group Q¹ and/or more than onereactive group Q². When more than one reactive groups Q¹ are present thegroups Q¹ may be the same or different, and when more than one reactivegroups Q² are present the groups Q² may be the same or different.

The present invention also relates to a compound, more particularly to alinker-construct, said compound comprising an alpha-end and anomega-end, the compound comprising on the alpha-end a functional groupQ¹ capable of reacting with a functional group F¹ present on abiomolecule and on the omega-end a functional group Q² capable ofreacting with a functional group F² present on a target molecule, thecompound further comprising a group according to formula (1), or a saltthereof, wherein said group according to formula (1) is as definedabove, and wherein the group according to formula (1), or a saltthereof, is situated in between said alpha-end and said omega-end of thecompound.

Reactive group Q¹ is covalently bonded to the alpha-end of the compound,and reactive group Q² is covalently bonded to a omega-end of thecompound.

This compound according to the invention may also be referred to as alinker-construct. In the linker-construct according to the invention, areactive group Q² is covalently connected to a reactive group Q¹ via alinker, and said linker comprises a group according to formula (1), or asalt thereof, as defined above. When the linker-construct according tothe invention comprises a salt of the group according to formula (1),the salt is preferably a pharmaceutically acceptable salt.

The linker-construct according to the invention may comprise more thanone reactive group Q². Consequently, the linker may thus comprise e.g. athird (fourth, fifth, etc.)-end, which may be referred to as a psi, chi,phi, etc.-end, the third (fourth, fifth, etc.) end comprising a reactivegroup Q². Similarly, the linker-conjugate may comprise more than onereactive group Q¹.

The linker-construct according to the invention may therefore also bedenoted as (Q¹)_(y)-Sp-(Q²)_(z), wherein y is an integer in the range of1 to 10 and z is an integer in the range of 1 to 10.

The invention thus also relates to a linker-construct according to theformula:(Q¹)_(y)-Sp-(Q²)_(z),wherein:y is an integer in the range of 1 to 10;z is an integer in the range of 1 to 10;Q¹ is a reactive group capable of reacting with a functional group F¹present on a biomolecule;Q² is a reactive group capable of reacting with a functional group F²present on a target molecule;Sp is a spacer moiety, wherein a spacer moiety is defined as a moietythat spaces (i.e. provides a certain distance between) and covalentlylinks reactive group Q¹ and reactive group Q²; andwherein said spacer moiety comprises a group according to Formula (1) ora salt thereof, wherein the group according to Formula (1) is as definedabove.

Preferably, y is 1, 2, 3 or 4, more preferably y is 1 or 2 and mostpreferably, y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferablyz is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, yet even morepreferably z is 1 or 2 and most preferably z is 1. More preferably, y is1 or 2, preferably 1, and z is 1, 2, 3 or 4, yet even more preferably yis 1 or 2, preferably 1, and z is 1, 2 or 3, yet even more preferably yis 1 or 2, preferably 1, and z is 1 or 2, and most preferably y is 1 andz is 1. In a preferred embodiment, the linker-construct is according tothe formula Q¹-Sp-(Q²)₄, Q¹-Sp-(Q²)₃, Q¹-Sp-(Q²)₂ or Q¹-Sp-Q².

The linker-construct according to the invention comprises a groupaccording to formula (1) as defined above, or a salt thereof. In apreferred embodiment, the linker-construct according to the inventioncomprises a group according to formula (1) wherein a is 0, or a saltthereof. In this embodiment, the linker-construct thus comprises a groupaccording to formula (2) or a salt thereof:

wherein R¹ is as defined above.

In another preferred embodiment, the linker-construct according to theinvention comprises a group according to formula (1) wherein a is 1, ora salt thereof. In this embodiment, the linker-construct thus comprisesa group according to formula (3) or a salt thereof:

wherein R¹ is as defined above.

In the linker-construct according to the invention, R¹ and preferredembodiments of R¹ are as defined above. Furthermore, reactive group Q¹and spacer moiety Sp, as well as preferred embodiments thereof, are asdefined above for the linker-conjugate according to the invention.

More particular, the invention relates to a compound according to theformula:(Q¹)_(y)-(Z^(w))-Sp-(Z^(x))-(Q²)_(z),wherein:y is an integer in the range of 1 to 10;z is an integer in the range of 1 to 10;Q¹ is a reactive group capable of reacting with a functional group F¹present on a biomolecule;Q² is a reactive group capable of reacting with a functional group F²present on a biomolecule;Sp is a spacer moiety, wherein a spacer moiety is defined as a moietythat spaces (i.e. provides a certain distance between) and covalentlylinks reactive groups Q¹ and Q²;Z^(w) is a connecting group connecting reactive group Q¹ to said spacermoiety;Z^(x) is a connecting group connecting reactive group Q² to said spacermoiety; and wherein said spacer moiety comprises a group according toFormula (1) or a salt thereof, wherein the group according to Formula(1) is as defined above.

In a preferred embodiment, a in the group according to formula (1) is 0.In another preferred embodiment, a in the group according to formula (1)is 1.

Preferred embodiments for y and z are as defined above for(Q¹)_(y)-Sp-(Q²)_(z). It is further preferred that the compound isaccording to the formula Q¹-(Z^(w))-Sp-(Z^(x))-(Q²)₄, Q-(Z^(w))—Sp-(Z^(x))-(Q²)₃, Q¹-(Z^(w))—Sp-(Z^(x))-(Q²)₂ orQ¹-(Z^(w))-Sp-(Z^(x))-Q², more preferably Q¹-(Z^(w))-Sp-(Z^(x))-(Q²)₂ orQ¹-(Z^(w))-Sp-(Z^(x))-Q² and most preferably Q¹-(Z^(w))-Sp-(Z^(x))-Q²,wherein Z and Z^(x) are as defined above.

In the linker compound according to the invention, Z and Z^(x) arepreferably independently selected from the group consisting of —O—, —S—,—NR²—, —N═N—, —C(O)—, —C(O)—NR²—, —O—C(O)—, —O—C(O)—O—, —O—C(O)—NR²,—NR₂—C(O)—, —NR²—C(O)—O—, —NR²—C(O)—NR²—, —S—C(O)—, —S—C(O)—O—,—S—C(O)—NR²—, —S(O)—, —S(O)₂—, —O—S(O)₂—, —O—S(O)₂—O—, —O—S(O)₂—NR²—,—O—S(O)—, —O—S(O)—O—, —O—S(O)—NR²—, —O—NR²—C(O)—, —O—NR²—C(O)—O—,—O—NR²—C(O)—NR²—, —NR²—O—C(O)—, —NR²—O—C(O)—O—, —NR²—O—C(O)—NR²—,—O—NR²—C(S)—, —O—NR²—C(S)—O—, —O—NR²—C(S)—NR²—, —NR²—O—C(S)—,—NR²—O—C(S)—O—, —NR²—O—C(S)—NR²—, —O—C(S)—, —O—C(S)—O—, —O—C(S)—NR²—,—NR²—C(S)—, —NR²—C(S)—O—, —NR²—C(S)—NR²—, —S—S(O)₂—, —S—S(O)₂—O—,—S—S(O)₂—NR²—, —NR²—O—S(O)—, —NR²—O—S(O)—O—, —NR²—O—S(O)—NR²—,—NR²—O—S(O)₂—, —NR²—S(O)₂—, —NR²—O—S(O)₂—NR²—, —O—NR²—S(O)—,—O—NR²—S(O)—O—, —O—NR²—S(O)—NR²—, —O—NR²—S(O)₂—O—, —O—NR²—S(O)₂—NR²—,—O—NR²—S(O)₂—, —O—P(O)(R²)₂—, —S—P(O)(R²)₂—, —NR²—P(O)(R²)₂— andcombinations of two or more thereof, wherein R² is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

Preferred embodiments for Q¹ are as defined above.

In the linker-construct according to the invention, Q² is a reactivegroup capable of reacting with a functional group F² present on a targetmolecule. Reactive groups Q² capable of reacting with such a functionalgroup F² are known to a person skilled in the art. In a preferredembodiment, Q² is a reactive group selected from the group consistingof, optionally substituted, N-maleimidyl groups, halogenatedN-alkylamido groups, sulfonyloxy N-alkylamido groups, ester groups,carbonate groups, sulfonyl halide groups, thiol groups or derivativesthereof, alkenyl groups, alkynyl groups, (hetero)cycloalkynyl groups,bicyclo[6.1.0]non-4-yn-9-yl] groups, cycloalkenyl groups, tetrazinylgroups, azido groups, phosphine groups, nitrile oxide groups, nitronegroups, nitrile imine groups, diazo groups, ketone groups,(O-alkyl)hydroxylamino groups, hydrazine groups, halogenatedN-maleimidyl groups, 1,1-bis(sulfonylmethyl)-methylcarbonyl groups orelimination derivatives thereof, carbonyl halide groups and allenamidegroups, —[C(R¹⁷)₂C(R¹⁷)₂O]_(q)—R¹⁷, wherein q is in the range of 1 to200, —CN, —NCV, —VCN, —VR¹⁷, —N(R¹⁷)₂, —⁺N(R¹⁷)₃—C(V)N(R¹⁷)₂,—C(R¹⁷)₂VR¹⁷, —C(V)R¹⁷, —C(V)VR¹⁷, —S(O)R¹⁷, —S(O)₂R¹⁷, —S(O)OR¹⁷,—S(O)₂OR¹⁷, —S(O)N(R¹⁷)₂, —S(O)₂N(R¹⁷)₂, —OS(O)R¹⁷, —OS(O)₂R¹⁷,—OS(O)OR¹⁷, —OS(O)₂OR¹⁷, —P(O)(R¹⁷)(OR¹⁷), —P(O)(OR¹⁷)₂, —OP(O)(OR¹⁷)₂,—Si(R¹⁷)₃, —VC(V)R¹⁷, —VC(V)VR¹⁷, —VC(V)N(R¹⁷)₂, —N(R¹⁷)C(V)R¹⁷,N(R¹⁷)C(V)VR¹⁷ and —N(R¹⁷)C(V)N(R¹⁷)₂, wherein V is O or S and whereinR¹⁷ is independently selected from the group consisting of hydrogen,halogen, C₁-C₂₄ alkyl groups, C₆-C₂₄ (hetero)aryl groups, C₇-C₂₄alkyl(hetero)aryl groups and C₇-C₂₄ (hetero)arylalkyl groups.

In a further preferred embodiment, Q² is according to formula (9a),(9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m),(9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y),(9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi),(9zj), (9zk), (9zl), (9zm) or (9zn), wherein (9a), (9b), (9c), (9d),(9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p),(9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za),(9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk),(9zl), (9zm), (9zn) and preferred embodiments thereof, are as definedabove. In this embodiment it is further preferred that Q² is accordingto formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s),(9t), (9zh), (9r), (9zl), (9zm) or (9zn), more preferably according toformula (9a), (9n), (9o), (9p), (9q), (9t), (9zh) or (9s), and even morepreferably Q² is according to formula (9a), (9p), (9q), (9n), (9t),(9zh), or (9o), and preferred embodiments thereof, as defined above.Most preferably, Q² is according to formula (9a), (9p), (9q), (9n),(9t), (9zh) or (9o), and preferred embodiments thereof, as definedabove.

In another further preferred embodiment, Q² is selected from the groupconsisting of —[C(R¹⁷)₂C(R¹⁷)₂O]_(q)—R¹⁷, wherein q is in the range of 1to 200, —CN, —NCV, —VCN, —VR¹⁷, —N(R¹⁷)₂, —⁺N(R¹⁷)₃, —C(V)N(R¹⁷)₂,—C(R¹⁷)₂VR¹⁷, —C(V)R¹⁷, —C(V) VR¹⁷, —S(O)R¹⁷, —S(O)₂R¹⁷, —S(O)OR¹⁷,—S(O)₂OR¹⁷, —S(O)N(R¹⁷)₂, —S(O)₂N(R¹⁷)₂, —OS(O)R¹⁷, —OS(O)₂R¹⁷,—OS(O)OR¹⁷, —OS(O)₂OR¹⁷, —P(O)(R¹⁷)(OR¹⁷), —P(O)(OR¹⁷)₂, —OP(O)(OR¹⁷)₂,—Si(R¹⁷)₃, —VC(V)R¹⁷, —VC(V)VR¹⁷, —VC(V)N(R¹⁷)₂, —N(R¹⁷)C(V)R¹⁷,—N(R¹⁷)C(V)VR¹⁷ and —N(R¹⁷)C(V)N(R¹⁷)₂, wherein V and R¹⁷ are as definedabove. In this embodiment it is further preferred that Q² is selectedfrom the group consisting of —OR¹⁷, —SR¹⁷, —N(R¹⁷)₂, —⁺N(R¹⁷)₃,—C(O)N(R¹⁷)₂, —C(R¹⁷)₂₀R¹⁷, —C(O)R¹⁷, —C(O)OR¹ 7, —S(O)R¹⁷, —S(O)₂R¹⁷,—S(O)OR¹⁷, —S(O)₂R¹⁷, —S(O)N(R¹⁷)₂, —S(O)₂N(R¹⁷)₂, —OS(O)R¹⁷,—OS(O)₂R¹⁷, —OS(O)OR¹⁷, —OS(O)₂OR¹⁷, —P(O)(R¹⁷)(OR¹⁷), —P(O)(OR¹⁷)₂,—OP(O)(O R¹⁷)₂, —Si(R¹⁷)₃, —OC(O)R¹⁷, —OC(O)OR¹⁷, —OC(O)N(R¹⁷)₂,—N(R¹⁷)C(O)R¹⁷, —N(R¹⁷)C(O)OR¹⁷ and —N(R¹⁷)C(O)N(R¹⁷)₂, wherein R¹⁷ isas defined above.

In a specific embodiment of the linker-construct according to theinvention, Q¹ is according to formula (9a), (9b), (9c), (9d), (9e),(9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q),(9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb),(9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl),(9zm) or (9zn), wherein (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h),(9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t),(9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze),(9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm), (9zn) andpreferred embodiments thereof, are as defined above; and spacer moietySp is selected from the group consisting of linear or branched C₁-C₂₀alkylene groups, the alkylene groups being optionally substituted andoptionally interrupted by one or more heteroatoms selected from thegroup consisting of O, S and NR³, wherein R³ is independently selectedfrom the group consisting of hydrogen and C₁-C₄ alkyl groups; whereinthe spacer moiety Sp is interrupted by one or more groups according toformula (1) or a salt thereof, and wherein (1) is as defined above.

In a preferred embodiment, said spacer moiety Sp is interrupted by oneor more groups according to formula (2), or a salt thereof, wherein (2)is as defined above. In another preferred embodiment, said spacer moietySp is interrupted by one or more groups according to formula (3), or asalt thereof, wherein (3) is as defined above.

In a further preferred embodiment, Q¹ is according to formula (9a),(9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9zh),(9zk), (9r), (9zl), (9zm) or (9zn). In an even further preferredembodiment, Q¹ is according to formula (9a), (9n), (9o), (9p), (9q),(9t), (9zh) or (9s), and in a particularly preferred embodiment, Q¹ isaccording to formula (9a), (9p), (9q), (9n), (9t), (9zh), (9zk) or (9o),and preferred embodiments thereof, as defined above. Most preferably Q¹is according to formula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o), andpreferred embodiments thereof, as defined above.

Process for the Preparation of a Linker-Conjugate

The present invention also relates to a process for the preparation of alinker-conjugate according to the invention. In particular, theinvention relates to a process for the preparation of a linker-conjugateaccording to the invention, the process comprising the step of reactinga functional group Q² of a linker-construct with a functional group F²of a target molecule, wherein said linker-construct is a compoundcomprising an alpha-end and an omega-end, the compound comprising on thealpha-end a functional group Q¹ capable of reacting with a functionalgroup F¹ present on a biomolecule and on the omega-end a functionalgroup Q² capable of reacting with a functional group F² present on saidtarget molecule, the compound further comprising a group according toformula (1), or a salt thereof, wherein said group according to formula(1) is as defined above, and wherein the group according to formula (1),or the salt thereof, is situated in between said alpha-end and saidomega-end of the compound.

The linker-construct and preferred embodiments thereof, includingpreferred embodiments of Q¹, Q² and target molecule D, are described indetail above.

In a preferred embodiment of the process for the preparation of alinker-conjugate, the linker-construct is according to(Q¹)_(y)-Sp-(Q²)_(z) as defined above. In a further preferred embodimentof the process for the preparation of a linker-conjugate, thelinker-construct is according to (Q¹)_(y)-(Z^(w))—Sp-(Z^(x))-(Q²)_(z) asdefined above.

The invention further relates to the use of a sulfamide linker-constructaccording to the invention in a bioconjugation process. Thelinker-construct according to the invention, and preferred embodimentstherefore, are described in detail above. The invention particularlyrelates to the use of a linker-construct according to formula(Q¹)-Sp-(Q²)_(z) in a bioconjugation process, and to the use of alinker-construct according to formula(Q¹)_(y)-(Z^(w))—Sp-(Z^(x))-(Q²)_(z) in a bioconjugation process.

The invention also relates to the use of a linker-conjugate according tothe invention in a bioconjugation process. The linker-conjugateaccording to the invention, and preferred embodiments therefore, aredescribed in detail above. The invention particularly relates to the useof a linker-conjugate according to formula (4a), (4b), (4c), (4d), (6a),(6b), (7a) or (7b) in a bioconjugation process.

Bioconjugate

A bioconjugate is herein defined as a compound wherein a biomolecule iscovalently connected to a target molecule via a linker. A bioconjugatecomprises one or more biomolecules and/or one or more target molecules.The linker may comprise one or more spacer moieties. The bioconjugateaccording to the invention is conveniently prepared by the process forpreparation of a bioconjugate according to the invention, wherein thelinker-conjugate comprising reactive group Q¹ is conjugated to abiomolecule comprising reactive group F¹. In this conjugation reaction,reactive groups Q¹ and F¹ react with each other to form a linker moiety,which joins the linker-conjugate with the biomolecule. All preferredembodiments described herein for the linker-conjugate and thebiomolecule thus equally apply to the bioconjugate according to theinvention, except for all said for Q¹ and F¹, wherein the bioconjugateaccording to the invention contains the reaction product of Q¹ and F¹ asdefined herein.

The invention also relates to a compound, the compound comprising analpha-end and an omega-end, the compound comprising on the alpha-end abiomolecule and on the omega-end a target molecule, the compound furthercomprising a group according to formula (1) or a salt thereof:

wherein:

-   -   a is 0 or 1; and    -   R¹ is selected from the group consisting of hydrogen, C₁-C₂₄        alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl        groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups, the C₁-C₂₄ alkyl groups, C₃-C₂₄        cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups; or    -   R¹ is a target molecule D, wherein the target molecule is        optionally connected to N via a spacer moiety;        and wherein the group according to formula (1), or the salt        thereof, is situated in between said alpha-end and said        omega-end.

In a preferred embodiment, the compound further comprises a moiety thatis obtainable by a cycloaddition reaction, preferably a 1,3-dipolarcycloaddition reaction, most preferably a 1,2,3-triazole ring, which islocated between said alpha-end and said group according to formula (1).Herein, the compound thus comprises, when view for the alpha end to theomega end, a biomolecule, a moiety that is obtainable by a cycloadditionreaction, a group according to formula (1) and a target molecule.

In a further preferred embodiment, the target molecule is hydrophobic asdefined hereinabove.

This compound according to the invention may also be referred to as abioconjugate. When the bioconjugate according to the invention comprisesa salt of the group according to formula (1), the salt is preferably apharmaceutically acceptable salt.

The biomolecule is covalently attached to the alpha-end, and the targetmolecule is covalently attached to the omega-end of the bioconjugateaccording to the invention.

The bioconjugate according to the invention may comprise more than onetarget molecule. Similarly, the bioconjugate may comprise more than onebiomolecule.

Biomolecule B and target molecule D, and preferred embodiments thereof,are described in more detail above. Preferred embodiments for D in thebioconjugate according to the invention correspond to preferredembodiments of D in the linker-conjugate according to the invention aswere described in more detail above. Preferred embodiments for thelinker (8a) or (8b) in the bioconjugate according to the inventioncorrespond to preferred embodiments of the linker in thelinker-conjugate according to the invention, as were described in moredetail above.

In the bioconjugate according to the invention, biomolecule B ispreferably selected from the group consisting of proteins (includingglycoproteins and antibodies), polypeptides, peptides, glycans, lipids,nucleic acids, oligonucleotides, polysaccharides, oligosaccharides,enzymes, hormones, amino acids and monosaccharides. More preferably,biomolecule B is selected from the group consisting of proteins(including glycoproteins and antibodies), polypeptides, peptides,glycans, nucleic acids, oligonucleotides, polysaccharides,oligosaccharides and enzymes. Most preferably, biomolecule B is selectedfrom the group consisting of proteins, including glycoproteins andantibodies, polypeptides, peptides and glycans.

The bioconjugate according to the invention may also be defined as abioconjugate wherein a biomolecule is conjugated to a target moleculevia a spacer-moiety, wherein the spacer-moiety comprises a groupaccording to formula (1), or a salt thereof, wherein the group accordingto formula (1) is as defined above.

The bioconjugate according to the invention may also be denoted as(B)_(y)—Sp-(D)_(z), wherein y is an integer in the range of 1 to 10 andz is an integer in the range of 1 to 10.

The invention thus also relates to a bioconjugate according to theformula:(B)_(y)-Sp-(D)_(z),wherein:y is an integer in the range of 1 to 10;z is an integer in the range of 1 to 10;B is a biomolecule;D is a target molecule;Sp is a spacer moiety, wherein a spacer moiety is defined as a moietythat spaces (i.e. provides a certain distance between) and covalentlylinks biomolecule B and target molecule D; andwherein said spacer moiety comprises a group according to formula (1) ora salt thereof, wherein the group according to formula (1) is as definedabove.

In a preferred embodiment, said spacer moiety further comprises a moietythat is obtainable by a cycloaddition reaction, preferably a 1,3-dipolarcycloaddition reaction, most preferably a 1,2,3-triazole ring, which islocated between B and said group according to formula (1).

Preferably, y is 1, 2, 3 or 4, more preferably y is 1 or 2 and mostpreferably, y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferablyz is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, yet even morepreferably z is 1 or 2 and most preferably z is 1. More preferably, y is1 or 2, preferably 1, and z is 1, 2, 3 or 4, yet even more preferably yis 1 or 2, preferably 1, and z is 1, 2 or 3, yet even more preferably yis 1 or 2, preferably 1, and z is 1 or 2, and most preferably y is 1 andz is 1. In a preferred embodiment, the bioconjugate is according to theformula B-Sp-(D)₄, B-Sp-(D)₃, B-Sp-(D)₂ or B-Sp-D.

As described above, the bioconjugate according to the inventioncomprises a group according to formula (1) as defined above, or a saltthereof. In a preferred embodiment, the bioconjugate comprises a groupaccording to formula (1) wherein a is 0, or a salt thereof. In thisembodiment, the bioconjugate thus comprises a group according to formula(2) or a salt thereof, wherein (2) is as defined above.

In another preferred embodiment, the bioconjugate comprises a groupaccording to formula (1) wherein a is 1, or a salt thereof. In thisembodiment, the bioconjugate thus comprises a group according to formula(3) or a salt thereof, wherein (3) is as defined above.

In the bioconjugate according to the invention, R¹, spacer moiety Sp, aswell as preferred embodiments of R¹ and Sp, are as defined above for thelinker-conjugate according to the invention.

In a preferred embodiment, the bioconjugate is according to formula (5a)or (5b), or a salt thereof:

wherein a, b, c, d, e, f, g, h, i, D, Sp¹, Sp², Sp³, Sp⁴, Z¹, Z², Z³ andR¹, and preferred embodiments thereof, are as defined above forlinker-conjugate (4a) and (4b); andh is 0 or 1; Z³ is a connecting group that connects B to Sp³, Z¹, Sp², Oor C(O); and B is a biomolecule.

Preferably, h is 1.

Preferred embodiments of biomolecule B are as defined above.

When the bioconjugate according to the invention is a salt of (5a) or(5b), the salt is preferably a pharmaceutically acceptable salt.

Z³ is a connecting group. As described above, the term “connectinggroup” herein refers to the structural element connecting one part of acompound and another part of the same compound. Typically, abioconjugate is prepared via reaction of a reactive group Q¹ present ina linker-conjugate with a functional group F¹ present in a biomolecule.As will be understood by the person skilled in the art, the nature ofconnecting group Z³ depends on the type of organic reaction that wasused to establish the connection between a biomolecule and alinker-conjugate. In other words, the nature of Z³ depends on the natureof reactive group Q¹ of the linker-conjugate and the nature offunctional group F¹ in the biomolecule. Since there is a large number ofdifferent chemical reactions available for establishing the connectionbetween a biomolecule and a linker-conjugate, consequently there is alarge number of possibilities for Z³.

Several examples of suitable combinations of F¹ and Q¹, and ofconnecting group Z³ that will be present in a bioconjugate when alinker-conjugate comprising Q¹ is conjugated to a biomolecule comprisinga complementary functional group F¹, are shown in FIG. 22.

When F¹ is for example a thiol group, complementary groups Q¹ includeN-maleimidyl groups and alkenyl groups, and the corresponding connectinggroups Z³ are as shown in FIG. 22. When F¹ is a thiol group,complementary groups Q¹ also include allenamide groups.

When F¹ is for example an amino group, complementary groups Q¹ includeketone groups, activated ester groups and azido groups, and thecorresponding connecting groups Z³ are as shown in FIG. 22.

When F¹ is for example a ketone group, complementary groups Q¹ include(O-alkyl)hydroxylamino groups and hydrazine groups, and thecorresponding connecting groups Z³ are as shown in FIG. 22.

When F¹ is for example an alkynyl group, complementary groups Q¹ includeazido groups, and the corresponding connecting group Z³ is as shown inFIG. 22.

When F¹ is for example an azido group, complementary groups Q¹ includealkynyl groups, and the corresponding connecting group Z³ is as shown inFIG. 22.

When F¹ is for example a cyclopropenyl group, a trans-cyclooctene groupor a cyclooctyne group, complementary groups Q¹ include tetrazinylgroups, and the corresponding connecting group Z³ is as shown in FIG.22. In these particular cases, Z³ is only an intermediate structure andwill expel N₂, thereby generating a dihydropyridazine (from the reactionwith alkene) or pyridazine (from the reaction with alkyne).

Additional suitable combinations of F¹ and Q¹, and the nature ofresulting connecting group Z³ are known to a person skilled in the art,and are e.g. described in G. T. Hermanson, “Bioconjugate Techniques”,Elsevier, 3^(rd) Ed. 2013 (ISBN:978-0-12-382239-0), in particular inChapter 3, pages 229-258, incorporated by reference. A list ofcomplementary reactive groups suitable for bioconjugation processes isdisclosed in Table 3.1, pages 230-232 of Chapter 3 of G. T. Hermanson,“Bioconjugate Techniques”, Elsevier, 3^(rd) Ed. 2013(ISBN:978-0-12-382239-0), and the content of this Table is expresslyincorporated by reference herein.

In the bioconjugate according to (5a) and (5b), it is preferred that atleast one of Z³, Sp³, Z¹ and Sp² is present, i.e. at least one of h, g,d and c is not 0. It is also preferred that at least one of Sp¹, Z² andSp⁴ is present, i.e. that at least one of b, e and i is not 0. Morepreferably, at least one of Z³, Sp³, Z¹ and Sp² is present and at leastone of Sp¹, Z² and Sp⁴ is present, i.e. it is preferred that at leastone of b, e and i is not 0 and at least one of h, g, d and c is not 0.

Process for the Preparation of a Bioconjugate

The present invention also relates to a process for the preparation of abioconjugate, the process comprising the step of reacting a reactivegroup Q¹ of a linker-conjugate according to the invention with afunctional group F¹ of a biomolecule. The linker-conjugate according tothe invention, and preferred embodiments thereof, are described in moredetail above.

FIG. 1 shows the general concept of conjugation of biomolecules: abiomolecule of interest (BOI) comprising one or more functional groupsF¹ is incubated with (excess of) a target molecule D (also referred toas molecule of interest or MOI) covalently attached to a reactive groupQ¹ via a specific linker. In the process of bioconjugation, a chemicalreaction between F¹ and Q¹ takes place, thereby forming a bioconjugatecomprising a covalent bond between the BOI and the MOI. The BOI may e.g.be a peptide/protein, a glycan or a nucleic acid. In the processaccording to the invention, the linker is a sulfamide linker.

The present invention thus relates to a process for the preparation of abioconjugate, the process comprising the step of reacting a reactivegroup Q¹ of a linker-conjugate with a functional group F¹ of abiomolecule, wherein the linker-conjugate is a compound comprising analpha-end and an omega-end, the compound comprising on the alpha-end areactive group Q¹ capable of reacting with a functional group F¹ presenton the biomolecule and on the omega-end a target molecule, the compoundfurther comprising a group according to formula (1) or a salt thereof:

wherein:

-   -   a is 0 or 1; and    -   R¹ is selected from the group consisting of hydrogen, C₁-C₂₄        alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl        groups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄        (hetero)arylalkyl groups, the C₁-C₂₄ alkyl groups, C₃-C₂₄        cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄        alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups        optionally substituted and optionally interrupted by one or more        heteroatoms selected from O, S and NR³ wherein R³ is        independently selected from the group consisting of hydrogen and        C₁-C₄ alkyl groups, or R¹ is a target molecule D, wherein the        target molecule is optionally connected to N via a spacer        moiety;        and wherein the group according to formula (1), or the salt        thereof, is situated in between said alpha-end and said        omega-end of the linker conjugate.

In a preferred embodiment, the invention concerns a process for thepreparation of a bioconjugate via a cycloaddition, such as a(4+2)-cycloaddition (e.g. a Diels-Alder reaction) or a(3+2)-cycloaddition (e.g. a 1,3-dipolar cycloaddition). Preferably, theconjugation is the Diels-Alder reaction or the 1,3-dipolarcycloaddition. The preferred Diels-Alder reaction is theinverse-electron demand Diels-Alder cycloaddition. In another preferredembodiment, the 1,3-dipolar cycloaddition is used, more preferably thealkyne-azide cycloaddition, and most preferably wherein Q¹ is orcomprises an alkyne group and F¹ is an azido group. Cycloadditions, suchas Diels-Alder reactions and 1,3-dipolar cycloadditions are known in theart, and the skilled person knowns how to perform them. In a furtherpreferred embodiment, the invention concerns a process for thepreparation of a bioconjugate, wherein the target molecule ishydrophobic (i.e. weakly soluble in water), most preferably wherein thetarget molecule has a water solubility of at most 0.1% (w/w) in water(20° C. and 100 kPa). In an especially preferred embodiment, theinvention concerns a process for the preparation of a bioconjugate viacycloaddition, preferably a 1,3-dipolar cycloaddition, more preferablythe alkyne-azide cycloaddition, and most preferably wherein Q¹ is orcomprises an alkyne group and F¹ is an azido group, and wherein thetarget molecule is hydrophobic, most preferably wherein the targetmolecule has a water solubility of at most 0.1% (w/w) in water (20° C.and 100 kPa).

In the process according to the invention, Q¹ reacts with F¹, forming acovalent connection between the biomolecule and the linker-moiety.Complementary reactive groups Q¹ and functional groups F¹ are describedin more detail above and below.

In a preferred embodiment of the process according to the invention, ais 0 in the group according to formula (1). In this embodiment, thelinker-conjugate thus comprises a group according to formula (2), asdefined above. In another preferred embodiment of the process accordingto the invention, a is 1 in the group according to formula (1). In thisembodiment, the linker-conjugate thus comprises a group according toformula (3), as defined above.

Biomolecules are described in more detail above. Preferably, in theprocess according to the invention the biomolecule is selected from thegroup consisting of proteins (including glycoproteins and antibodies),polypeptides, peptides, glycans, lipids, nucleic acids,oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones,amino acids and monosaccharides. More preferably, biomolecule B isselected from the group consisting of proteins (including glycoproteinsand antibodies), polypeptides, peptides, glycans, nucleic acids,oligonucleotides, polysaccharides, oligosaccharides and enzymes. Mostpreferably, biomolecule B is selected from the group consisting ofproteins, including glycoproteins and antibodies, polypeptides, peptidesand glycans.

Target molecules are described in more detail above. In a preferredembodiment of the process according to the invention, the targetmolecule is selected from the group consisting of an active substance, areporter molecule, a polymer, a solid surface, a hydrogel, ananoparticle, a microparticle and a biomolecule. Active substances,reporter molecules, polymers, solid surfaces, hydrogels, nanoparticlesand microparticles are described in detail above, as are their preferredembodiments. In view of the significantly improved water solubility ofthe linker-conjugate when the sulfamide linker according to theinvention is employed, a preferred embodiment of the process for thepreparation of a bioconjugate employs a hydrophobic target molecule. Thehydrophobic target molecule in its unconjugated form typically has awater solubility of at most 1% (w/w), preferably at most 0.1% (w/w),most preferably at most 0.01% (w/w), determined at 20° C. and 100 kPa.

In the process according to the invention, it is preferred that reactivegroup Q¹ is selected from the group consisting of, optionallysubstituted, N-maleimidyl groups, halogenated N-alkylamido groups,sulfonyloxy N-alkylamido groups, ester groups, carbonate groups,sulfonyl halide groups, thiol groups or derivatives thereof, alkenylgroups, alkynyl groups, (hetero)cycloalkynyl groups,bicyclo[6.1.0]non-4-yn-9-yl]groups, cycloalkenyl groups, tetrazinylgroups, azido groups, phosphine groups, nitrile oxide groups, nitronegroups, nitrile imine groups, diazo groups, ketone groups,(O-alkyl)hydroxylamino groups, hydrazine groups, halogenatedN-maleimidyl groups, 1,1-bis(sulfonylmethyl)methylcarbonyl groups orelimination derivatives thereof, carbonyl halide groups and allenamidegroups.

In a further preferred embodiment, Q¹ is according to formula (9a),(9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m),(9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y),(9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi),(9zj), (9zk), (9zl), (9zm) or (9zn), wherein (9a), (9b), (9c), (9d),(9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p),(9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za),(9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk),(9zl), (9zm), (9zn) and preferred embodiments thereof, are as definedabove for the linker-conjugate according to the invention. Morepreferably, Q¹ is according to formula (9a), (9b), (9c), (9f), (9j),(9n), (9o), (9p), (9q), (9s), (9t), (9zh), (9r), (9zl), (9zm) or (9zn).Even more preferably, Q¹ is according to formula (9a), (9n), (9o), (9p),(9q), (9t), (9zh) or (9s), and most preferably, Q¹ is according toformula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o), and preferredembodiments thereof, as defined above.

In an especially preferred embodiment, Q¹ comprises an alkyne group,preferably selected from the alkynyl group as described above, thecycloalkenyl group as described above, the (hetero)cycloalkynyl group asdescribed above and a bicyclo[6.1.0]non-4-yn-9-yl] group, morepreferably Q¹ is selected from the formulae (9j), (9n), (9o), (9p), (9q)and (9zk), as defined above. Most preferably, Q¹ is abicyclo[6.1.0]non-4-yn-9-yl] group, preferably of formula (9q).

In a further preferred embodiment of the process according to theinvention, the linker-conjugate is according to formula (4a) or (4b), ora salt thereof:

wherein:a is independently 0 or 1;b is independently 0 or 1;c is 0 or 1;d is 0 or 1;e is 0 or 1;f is an integer in the range of 1 to 150;g is 0 or 1;i is 0 or 1;D is a target molecule;Q¹ is a reactive group capable of reacting with a functional group F¹present on a biomolecule;Sp¹ is a spacer moiety;Sp² is a spacer moiety;Sp³ is a spacer moiety;Sp⁴ is a spacer moiety;Z¹ is a connecting group that connects Q¹ or Sp³ to Sp², O or C(O) orN(R¹);Z² is a connecting group that connects D or Sp⁴ to Sp¹, N(R¹), O orC(O); andR¹ is selected from the group consisting of hydrogen, C₁-C₂₄ alkylgroups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups, the C₁-C₂₄alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups,C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groupsoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁-C₄ alkyl groups;orR¹ is D, -[(Sp¹)_(b)-(Z²)_(e)-(Sp⁴)_(i)-D] or-[(Sp²)_(c)-(Z¹)_(d)-(Sp³)_(g)-Q¹], wherein Sp¹, Sp², Sp³, Sp⁴, Z¹, Z²,D, Q¹, b, c, d, e, g and i are as defined above.

Sp¹, Sp², Sp³ and Sp⁴ are, independently, spacer moieties, in otherwords, Sp¹, Sp², Sp³ and Sp⁴ may differ from each other. Sp¹, Sp², Sp³and Sp⁴ may be present or absent (b, c, g and i are, independently, 0 or1). However, it is preferred that at least one of Sp¹, Sp², Sp³ and Sp⁴is present, i.e. it is preferred that at least one of b, c, g and i isnot 0.

If present, preferably Sp¹, Sp², Sp³ and Sp⁴ are independently selectedfrom the group consisting of linear or branched C₁-C₂₀₀ alkylene groups,C₂-C₂₀₀ alkenylene groups, C₂-C₂₀₀ alkynylene groups, C₃-C₂₀₀cycloalkylene groups, C₅-C₂₀₀ cycloalkenylene groups, C₈-C₂₀₀cycloalkynylene groups, C₇-C₂₀₀ alkylarylene groups, C₇-C₂₀₀arylalkylene groups, C₈-C₂₀₀ arylalkenylene groups and C₉-C₂₀₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted. When the alkylene groups,alkenylene groups, alkynylene groups, cycloalkylene groups,cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups,arylalkylene groups, arylalkenylene groups and arylalkynylene groups areinterrupted by one or more heteroatoms as defined above, it is preferredthat said groups are interrupted by one or more O-atoms, and/or by oneor more S—S groups.

More preferably, spacer moieties Sp¹, Sp², Sp³ and Sp⁴, if present, areindependently selected from the group consisting of linear or branchedC₁-C₁₀₀ alkylene groups, C₂-C₁₀₀ alkenylene groups, C₂-C₁₀₀ alkynylenegroups, C₃-C₁₀₀ cycloalkylene groups, C₅-C₁₀₀ cycloalkenylene groups,C₈-C₁₀ cycloalkynylene groups, C₇-C₁₀₀ alkylarylene groups, C₇-C₁₀₀arylalkylene groups, C₈-C₁₀₀ arylalkenylene groups and C₉-C₁₀₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

Even more preferably, spacer moieties Sp¹, Sp², Sp³ and Sp⁴, if present,are independently selected from the group consisting of linear orbranched C₁-C₅₀ alkylene groups, C₂-C₅₀ alkenylene groups, C₂-C₅₀alkynylene groups, C₃-C₅₀ cycloalkylene groups, C₅-C₅₀ cycloalkenylenegroups, C₈-C₅₀ cycloalkynylene groups, C₇-C₅₀ alkylarylene groups,C₇-C₅₀ arylalkylene groups, C₈-C₅₀ arylalkenylene groups and C₉-C₅₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

Yet even more preferably, spacer moieties Sp¹, Sp², Sp³ and Sp⁴, ifpresent, are independently selected from the group consisting of linearor branched C₁-C₂₀ alkylene groups, C₂-C₂₀ alkenylene groups, C₂-C₂₀alkynylene groups, C₃-C₂₀ cycloalkylene groups, C₅-C₂₀ cycloalkenylenegroups, C₈-C₂₀ cycloalkynylene groups, C₇-C₂₀ alkylarylene groups,C₇-C₂₀ arylalkylene groups, C₈-C₂₀ arylalkenylene groups and C₉-C₂₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

In these preferred embodiments it is further preferred that the alkylenegroups, alkenylene groups, alkynylene groups, cycloalkylene groups,cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups,arylalkylene groups, arylalkenylene groups and arylalkynylene groups areunsubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, preferably 0, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups, preferably hydrogen or methyl.

Most preferably, spacer moieties Sp¹, Sp², Sp³ and Sp⁴, if present, areindependently selected from the group consisting of linear or branchedC₁-C₂₀ alkylene groups, the alkylene groups being optionally substitutedand optionally interrupted by one or more heteroatoms selected from thegroup of O, S and NR³, wherein R³ is independently selected from thegroup consisting of hydrogen, C₁-C₂₄ alkyl groups, C₂-C₂₄ alkenylgroups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkyl groups, the alkylgroups, alkenyl groups, alkynyl groups and cycloalkyl groups beingoptionally substituted. In this embodiment, it is further preferred thatthe alkylene groups are unsubstituted and optionally interrupted by oneor more heteroatoms selected from the group of O, S and NR³, preferablyO and/or S—S, wherein R³ is independently selected from the groupconsisting of hydrogen and C₁-C₄ alkyl groups, preferably hydrogen ormethyl.

Particularly preferred spacer moieties Sp¹, Sp², Sp³ and Sp⁴ include—(CH₂)_(n)—, —(CH₂CH₂)_(n)—, —(CH₂CH₂O)_(n)—, —(OCH₂CH₂)_(n)—,—(CH₂CH₂O)_(n)CH₂CH₂—, —CH₂CH₂(OCH₂CH₂)_(n)—, —(CH₂CH₂CH₂O)_(n)—,—(OCH₂CH₂CH₂)_(n)—, —(CH₂CH₂CH₂O)_(n)CH₂CH₂CH₂— and—CH₂CH₂CH₂(OCH₂CH₂CH₂)_(n)—, wherein n is an integer in the range of 1to 50, preferably in the range of 1 to 40, more preferably in the rangeof 1 to 30, even more preferably in the range of 1 to 20 and yet evenmore preferably in the range of 1 to 15. More preferably n is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3, 4, 5, 6, 7 or 8, evenmore preferably 1, 2, 3, 4, 5 or 6, yet even more preferably 1, 2, 3 or4.

In another preferred embodiment of the process according to theinvention, in the linker-conjugates according to formula (4a) and (4b),spacer moieties Sp¹, Sp², Sp³ and/or Sp⁴, if present, comprise asequence of amino acids. Spacer-moieties comprising a sequence of aminoacids are known in the art, and may also be referred to as peptidelinkers. Examples include spacer-moieties comprising a Val-Cit moiety,e.g. val-cit-PABC, val-cit-PAB, Fmoc-val-cit-PAB, etc.

As described above, Z¹ and Z² are a connecting groups. In a preferredembodiment of the process according to the invention, Z¹ and Z² areindependently selected from the group consisting of —O—, —S—, —NR²—,—N═N—, —C(O)—, —C(O)NR²—, —O—C(O)—, —O—C(O)—O—, —O—C(O)—NR², —NR₂—C(O)—,—NR²—C(O)—O—, —NR²—C(O)—NR²—, —S—C(O)—, —S—C(O)—O—, —S—C(O)—NR²—,—S(O)—, —S(O)₂—, —O—S(O)₂—, —O—S(O)₂—O—, —O—S(O)₂—NR²—, —O—S(O)—,—O—S(O)—O—, —O—S(O)—NR²—, —O—NR²—C(O)—, —O—NR²—C(O)—O—,—O—NR²—C(O)—NR²—, —NR²—O—C(O)—, —NR²—O—C(O)—O—, —NR²—O—C(O)—NR²—,—O—NR²—C(S)—, —O—NR²—C(S)—O—, —O—NR²—C(S)—NR²—, —NR²—O—C(S)—,—NR²—O—C(S)—O—, —NR²—O—C(S)—NR²—, —O—C(S)—, —O—C(S)—O—, —O—C(S)—NR²—,—NR²—C(S)—, —NR²—C(S)—O—, —NR²—C(S)—NR²—, —S—S(O)₂—, —S—S(O)₂—O—,—S—S(O)₂—NR²—, —NR²—O—S(O)—, —NR²—O—S(O)—O—, —NR²⁻O—S(O)—NR²—,—NR²—O—S(O)₂—, —NR²—O—S(O)₂—O—, —NR²—O—S(O)₂—NR²—, —O—NR²—S(O)—,—O—NR²—S(O)—O—, —O—NR²—S(O)—NR²—, —O—NR²—S(O)₂—O—, —O—NR²—S(O)₂—NR²—,—O—NR²—S(O)₂—, —O—P(O)(R²)₂—, —S—P(O)(R²)₂—, —NR²—P(O)(R²)₂— andcombinations of two or more thereof, wherein R² is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.

In a particularly preferred process according to the invention, Sp¹,Sp², Sp³ and Sp⁴, if present, are independently selected from the groupconsisting of linear or branched C₁-C₂₀ alkylene groups, the alkylenegroups being optionally substituted and optionally interrupted by one ormore heteroatoms selected from the group consisting of O, S and NR³,wherein R³ is independently selected from the group consisting ofhydrogen and C₁-C₄ alkyl groups, and wherein Q¹ is according to formula(9a), (9p), (9q), (9n), (9t), (9zh) or (9o):

wherein:R¹⁰ is hydrogen or a (thio)ester group; andR¹⁸ is selected from the group consisting of, optionally substituted,C₁-C₁₂ alkyl groups and C₄-C₁₂ (hetero)aryl groups.

As described above, in the process for the preparation of abioconjugate, a reactive group Q¹ that is present in a linker-conjugateis reacted with a functional group F¹ that is present in a biomolecule.In the process according to the invention, more than one functionalgroup may be present in the biomolecule. When two or more functionalgroups are present, said groups may be the same or different. Similarly,more than one reactive group may be present in the linker-conjugate.When two or more reactive groups are present, said groups may be thesame or different. In a preferred embodiment of the process according tothe invention, the linker-conjugate comprises one reactive group Q¹, andone or more target molecules D which may be the same or different. Thelinker-conjugate comprises for example 1, 2, 3, 4, 5 or 6, preferably 1,2, 3 or 4, more preferably 1, 2 or 3, even more preferably 1 or 2 targetmolecule D. In a particularly preferred embodiment the linker-conjugatecomprises 1 target molecule D.

In another particularly preferred embodiment, the linker-conjugatecomprises 2 target molecules D, which may be the same or different. Inanother preferred embodiment, the biomolecule comprises two or morefunctional groups F, which may be the same or different, and two or morefunctional groups react with a complementary reactive group Q of alinker-conjugate. For example a biomolecule comprising two functionalgroups F, i.e. F¹ and F², may react with two linker-conjugatescomprising a functional group Q¹, which may be the same or different, toform a bioconjugate.

Examples of a functional group F¹ in a biomolecule comprise an aminogroup, a thiol group, a carboxylic acid, an alcohol group, a carbonylgroup, a phosphate group, or an aromatic group. The functional group inthe biomolecule may be naturally present or may be placed in thebiomolecule by a specific technique, for example a (bio)chemical or agenetic technique. The functional group that is placed in thebiomolecule may be a functional group that is naturally present innature, or may be a functional group that is prepared by chemicalsynthesis, for example an azide, a terminal alkyne, a cyclopropenemoiety or a phosphine moiety. In view of the preferred mode ofconjugation by cycloaddition, it is preferred that F¹ is group capableof reacting in a cycloaddition, such as a diene, a dienophile, a1,3-dipole or a dipolarophile, preferably F¹ is selected from a1,3-dipole (typically an azido group, nitrone group, nitrile oxidegroup, nitrile imine group or diazo group) or a dipolarophile (typicallyan alkenyl or alkynyl group). Herein, F¹ is a 1,3-dipole when Q¹ is adipolarophile and F¹ is a dipolarophile when Q¹ is a 1,3-dipole, or F¹is a diene when Q¹ is a dienophile and F¹ is a dienophile when Q¹ is adiene. Most preferably, F¹ is a 1,3-dipole, preferably F¹ is orcomprises an azido group.

Several examples of a functional group that is placed into a biomoleculeare shown in FIG. 2. FIG. 2 shows several structures of derivatives ofUDP sugars of galactosamine, which may be modified with e.g. athiopropionyl group (11a), an azidoacetyl group (11b), or anazidodifluoroacetyl group (11c).

FIG. 3 schematically displays how any of the UDP-sugars 11a-c may beattached to a glycoprotein comprising a GlcNAc moiety 12 (e.g. amonoclonal antibody the glycan of which is trimmed by anendoglycosidase) under the action of a galactosyltransferase mutant or aGalNAc-transferase, thereby generating a (3-glycosidic 1-4 linkagebetween a GalNAc derivative and GlcNAc (compounds 13a-c, respectively).

Preferred examples of naturally present functional groups F¹ include athiol group and an amino group. Preferred examples of a functional groupthat is prepared by chemical synthesis for incorporation into thebiomolecule include a ketone group, a terminal alkyne group, an azidegroup, a cyclo(hetero)alkyne group, a cyclopropene group, or a tetrazinegroup.

As was described above, complementary reactive groups Q¹ and functionalgroups F¹ are known to a person skilled in the art, and several suitablecombinations of Q¹ and F¹ are described above, and shown in FIG. 22. Alist of complementary groups Q¹ and F¹ is disclosed in Table 3.1, pages230-232 of Chapter 3 of G. T. Hermanson, “Bioconjugate Techniques”,Elsevier, 3^(rd) Ed. 2013 (ISBN:978-0-12-382239-0), and the content ofthis Table is expressly incorporated by reference herein.

An embodiment of the process according to the invention is depicted inFIG. 4. FIG. 4 shows how a modified antibody 13a-c may undergo abioconjugation process by means of nucleophilic addition with maleimide(as for 13a, leading to thioether conjugate 14) or upon strain-promotedcycloaddition with a cyclooctyne reagent (as for 13b or 13c, leading totriazoles 15 or 16, respectively).

The invention further relates to a bioconjugate obtainable by theprocess according to the invention for the preparation of abioconjugate.

An advantage of the process according to the invention for thepreparation of a bioconjugate, and of the linker-conjugate and sulfamidelinker according to the invention is that conjugation efficiencyincreases in case a sulfamide linker is used instead of a typicalpolyethylene glycol (PEG) spacer. For example, as demonstrated inexample 58, a competition experiment involving incubation of trastuzumabcontaining a single engineered free cysteine with a stoichiometricmixture of maleimide 17 (comprising a comparative linker) and 18(comprising a sulfamide linker according to the invention) demonstratedthat conjugation efficiency of the sulfamide-containing maleimide 18 ishigher than with 17.

FIG. 5 shows the structure of linker-conjugates 17 and 18, wherein Q¹ isan N-maleimidyl group and D is a pyrene. Compound 18 is according to theinvention, whereas compound 17 is a comparative example. FIG. 16 showsthe synthesis of 17 and 18.

Likewise, conjugations of 19-35, depicted in FIGS. 6-9, withtrastuzumab-N₃ show that a conjugation process based onazide-cyclooctyne cycloaddition is invariably faster forsulfamide-containing linker-conjugates in comparison to the traditionalPEG-containing linker conjugates, and in most cases dramatically faster(see Table 1 and 2 and FIGS. 12-14).

Conjugates of 20, 21, 23, 26, 29, 33 and 35 with trastuzumab-N₃ areaccording to the invention, and conjugates of 19, 22, 24, 25, 27, 28,30, 31, 32 and 34 are comparative examples. Moreover, in severalinstances conjugations with PEG-based constructs do not reach fullconversion, even after prolonged incubation times, e.g.

FIGS. 12a, 12b, 13b and 14a .

TABLE 1 Conversion (%) Compound 30′ 60′ 90′ 120′ 240′ 960′ 19 (comp.)  0 0 n.d. n.d. 10 n.d. 20 80 90 n.d. 95 >95  n.d. 21 n.d. 60 n.d. n.d. 90n.d. 22 (comp.)  0  5 n.d. 10 30 n.d. 23 20 30 n.d.  70* >90* n.d. 24(comp.)  0 10 20 30 n.d. 80 25 (comp.) 20 20 30 60 n.d. 80 26 70 80 8595 n.d. 100  27 (comp.) 20 25 30 30 n.d. 30 28 (comp.) 30 40 60 65 n.d.65 29 60 70 80 85 n.d. 90 30 (comp.) 75 >90  n.d. >95  n.d. >95  31(comp.) 25 45 n.d. 60 n.d. 95 32 (comp.) 20 35 n.d. 70 n.d. 85 33 85 95n.d. >95  n.d. >95  34 (comp.) 25 50 n.d. 75 n.d. 95 35 95 >95 n.d. >95  n.d. >95  *Based on remaining starting trastuzumab-N₃. n.d. =not determined

TABLE 2 conversion (%) compound 30′ 60′ 120′ 240′ 3600′ 30 (comp.) 25 4065 95 95 31 (comp.)  5 10 15 50 50 32 (comp.) <5  5 10 80 80 33 25 40 6595 95 34 (comp.) <5 <5  5 85 85 35 80 90 95 >95  >95 

FIG. 12a shows the conjugation efficiency of BCN-pyrene derivativesconjugated via a sulfamide linker (compound 26) or short PEG linkers(compounds 24 or 25) with trastuzumab-N₃ (compound 13b).

FIG. 12b shows the conjugation efficiency of DIBAC-pyrene derivativesconjugated via a sulfamide linker (compound 29) or short PEG linkers(compounds 27 or 28) with trastuzumab-N₃ (compound 13b).

FIG. 13a shows the conjugation efficiency of BCN-maytansin derivativesconjugated via a sulfamide linker (compound 33) or short PEG linkers(compounds 30-32) with trastuzumab-N₃ (compound 13b).

FIG. 13b shows the conjugation efficiency of BCN-maytansin derivativesconjugated via a sulfamide linker (compounds 33) or short PEG linkers(compounds 30-32, with compound 30 overlapping with 33) withtrastuzumab-F₂-GalNAz (compound 13c).

FIG. 14a shows the conjugation efficiency of BCN-duocarmycin derivativesconjugated via a sulfamide linker (compound 35) or short PEG linker(compound 34) with trastuzumab-N₃ (compound 13b).

FIG. 14b shows the conjugation efficiency of BCN-duocarmycin derivativesconjugated via a sulfamide linker (compound 35) or short PEG linker(compound 34) with trastuzumab-F₂-GalNAz (compound 13c).

An additional advantage of a sulfamide group, in particular of anacylsulfamide or a carbamoylsulfamide group, is its high polarity, whichimparts a positive effect on the solubility of a linker comprising suchgroup, and on the construct as a whole, before, during and afterconjugation. The increased polarity of the sulfamide spacer becomesclear from Table 3 (graphically depicted in FIG. 11), which summarizesthe retention times of compounds 19-23 and 30-38 on RP-HPLC. In view ofthis increased polarity, conjugation with linker-conjugates containingthe sulfamide linker according to the invention are particularly suitedto conjugate hydrophobic target compounds to a biomolecule.

FIG. 11 shows the HPLC retention times of compounds 19-23 and 30-38 with0.1% TFA or in buffer pH 7.4.

FIG. 6 shows the structures of several compounds wherein abicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (also referred to as aBCN group) is connected to benzylamine (D) via a linker unit. Compounds20, 21 and 23 are according to the invention, whereas compounds 19 and22 are comparative examples. FIG. 17 shows the synthesis of 19-22 (seealso Examples 21-28) and FIG. 18 the synthesis of 23 (see also Examples29-31).

FIG. 7 shows the structures of several compounds wherein abicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (also referred to as aBCN group) or a dibenzoazocyclooctyne reactive group Q¹ (also referredto as a DIBAC group or DBCO group) is connected to pyrene via a linkerunit. Compounds 26 and 29 are according to the invention, whereascompounds 24, 25, 27 and 28 are comparative examples. FIG. 19 shows thesynthetic routes leading to compounds 24-26, and FIG. 20 shows thesynthetic routes leading to compounds 27-29.

FIG. 8 shows the structures of several compounds wherein abicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (also referred to as aBCN group) is connected to maytansin via a linker unit. Compound 33 isaccording to the invention, whereas compounds 30, 31 and 32 arecomparative examples.

FIG. 9 shows the structures of several compounds wherein abicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (also referred to as aBCN group) is connected to a Val-Cit-PABA-duocarmycin construct via alinker unit. Compound 35 is according to the invention, whereas compound34 is a comparative example.

FIG. 10 shows the structures of compounds 36-38 according to theinvention wherein a bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q¹ (alsoreferred to as a BCN group) is conjugated to Val-Cit-PABA-Ahx-maytansinvia a linker.

The synthesis of compounds 30-35 is described in examples 43-49 and thesynthetic routes for compounds 36-38 are graphically depicted in FIG. 21and described in examples 50-55.

TABLE 3 Retention times of compounds 19-23 and 30-38 measured byRP-HPLC. retention time (min) compound 0.1% TFA pH 7.4 19 (comp.) 11.411.4 20 11.6 9.6 21 11.6 9.2 22 (comp.) 12.7 12.6 23 10.6 6.9 30 (comp.)10.5 10.6 31 (comp.) 10.1 10.1 32 (comp.)  9.7 9.7 33 10.4 9.3 34(comp.) 11.5 11.5 35 11.6 10.0 36 10.4 9.3 37 11.2 10.5 38 11.1 9.5

As more polar compounds show reduced retention time, the lower valuesfor compounds 20, 21 and 23 with respect to compound 19 and 22 at pH 7.4(no effect is visible at low pH), clearly reflects the polarity ofsulfamide spacers in comparison to normal amide-linker constructs(compound 19). Methylated compound 22, although also a sulfamide in thestrict sense, does not display the enhanced polarity, due to the lack ofan acidic proton at the acylated nitrogen. The fact that no cleardifference in retention time is visible with 0.1% TFA (at which pH nodeprotonation will take place) is also an indication that the acidicproton plays a key role in defining polarity. The bissulfamide compound22 finally underlines that additional sulfamide units in a single linker(i.e. when f is 2 or more in the compounds and process according to theinvention) further increases the polarity. The latter observation hasalso facilitated the smooth conjugation of a BCN-construct bearing twolipophilic toxins (maytansins), as in bissulfamide compound 38 and 39,to an azido-mAb.

The high polarity of the sulfamides also has a positive impact in casehydrophobic moieties are imparted onto a biomolecule of interest, whichis known to require large amounts of organic cosolvent duringconjugation and/or induce aggregation of the bioconjugate. High levelsof cosolvent (up to 50% of DMA, propylene glycol, or DMSO) may induceprotein denaturation during the conjugation process and/or may requirespecial equipment in the manufacturing process. An example of thestability of the bioconjugate involves the highly polarbissulfamide-containing compound 38, which once conjugated totrastuzumab, displayed zero to negligible aggregation upon prolongedstorage, which in fact also applied to monosulfamide 37.

In view of this reduced tendency to aggregate, the sulfamide linkeraccording to the invention is particularly beneficial to be used toprepare bioconjugates wherein the reaction product of the conjugationreaction, i.e. reaction between reactive groups Q¹ and F¹, affords alinking moiety which is weakly water soluble. In an especially preferredembodiment, the conjugation is accomplished via a cycloaddition,preferably a 1,3-dipolar cycloaddition, more preferably alkyne-azidecycloaddition. As shown in the examples, any aggregation herein isbeneficially reduced using the sulfamide linker according to theinvention, which greatly improves the stability of the product byreducing the tendency to aggregate. Thus, the problem of aggregationassociated with the hydrophobic linking moieties in bioconjugates isefficiently solved by using the sulfamide linker according to theinvention in the spacer between the target molecule and the reactivegroup Q¹ in the linker-conjugate in the formation of the bioconjugate.

An additional advantage of a sulfamide linker according the invention,and its use in bioconjugation processes, is its ease of synthesis andhigh yields. As schematically depicted in FIG. 15, synthesis of acarbamoyl sulfamide spacer involves the reaction of a primary alcohol 40with the commercially available reagent CSI (chlorosulfonyl isocyanate),leading to an intermediate sulfonylchloride 41 that without work-up isreacted with an amine thereby affording the stable carbamoyl sulfamide42. The latter compound can be easily converted into an acylsulfamide incase R¹ is a tert-butyl, which upon acid treatment affords a primarysulfamide product 43. Similarly, in case R¹=benzyl, deprotection can beaffected with hydrogenation. The primary sulfamide 43 can beconveniently acylated with activated esters with common procedures togive acylsulfamide 44.

The ease of synthesis of sulfamide linkers according the invention, andtheir excellent performance in bioconjugation processes also becomesclear in the synthesis and utility of compounds 23 and 38, bothbissulfamide constructs that are readily generated by repetition of theevents summarized above, i.e. treatment of alcohol with CSI, followed byreaction with an aminoalcohol generates in return an alcohol that mayagain undergo the sequence of events, thereby generating a bissulfamide.Further repetition of the sequence generate tri-, tetra- and higheroligomers of sulfamide, depending on the number of repetitions.

The fact that the sulfamide linker in the final conjugate is short mayhave an additional advantage in the sense that it is known that inparticular long PEG spacers (e.g. PEG₂₄), needed to counterbalance thehydrophobic character of a target molecules such as a cytotoxin, mayhave a negative impact on pharmacokinetics of the final proteinconjugate, as is known for antibody-drug conjugates, in particular withhigh drug loading.

EXAMPLES Example 1 Synthesis ofα-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose 1-phosphate

α-2-Azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose 1-phosphate wasprepared from D-galactosamine according to procedures described forD-glucosamine in Linhardt et al., J. Org. Chem. 2012, 77, 1449-1456,incorporated by reference.

¹H-NMR (300 MHz, CD₃OD): δ (ppm) 5.69 (dd, J=7.2, 3.3 Hz, 1H), 5.43-5.42(m, 1H), 5.35 (dd, J=11.1, 3.3 Hz, 1H), 4.53 (t, J=7.2 Hz, 1H),4.21-4.13 (m, 1H), 4.07-4.00 (m, 1H), 3.82 (dt, J=10.8, 2.7 Hz, 1H),2.12 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H).

LRMS (ESI⁻) m/z calcd for C₁₂H₁₇N₃O₁₁P (M−H⁺)=410.06; found 410.00.

Example 2 Synthesis ofca-UDP-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose

α-2-Azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose 1-phosphate, asprepared in example 1, was attached to UMP according to Baisch et al.Bioorg. Med. Chem., 1997, 5, 383-391.

Thus, a solution of D-uridine-5′-monophosphate disodium salt (1.49 g,4.05 mmol) in H₂O (15 mL) was treated with DOWEX 50Wx8 (H⁺ form) for 30minutes and filtered.

The filtrate was stirred vigorously at room temperature whiletributylamine (0.97 mL, 4.05 mmol) was added dropwise. After 30 minutesof further stirring, the reaction mixture was lyophilized and furtherdried over P₂O₅ under vacuum for 5 h.

The resulting tributylammonium uridine-5′-monophosphate was dissolved indry DMF (25 mL) in an argon atmosphere. Carbonyl diimidazole (1.38 g,8.51 mmol) was added and the reaction mixture was stirred at r.t. for 30min. Next, dry MeOH (180 μL) was added and stirred for 15 min to removethe excess carbonyldiimidazole. The leftover MeOH was removed under highvacuum for 15 min. The resulting compound (2.0 g, 4.86 mmol) wasdissolved in dry DMF (25 mL) and added dropwise to the reaction mixture.The reaction was allowed to stir at rt for 2 d before concentration invacuo. The consumption of the imidazole-UMP intermediate was monitoredby MS. Gradient flash column chromatography (7:2:1→5:2:1 EtOAc:MeOH:H₂O)afforded α-UDP-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose (1.08 g,1.51 mmol, 37%).

¹H-NMR (300 MHz, D₂O): δ (ppm) 7.96 (d, J=8.0 Hz, 1H), 5.98-5.94 (m,2H), 5.81-5.79 (m, 1H), 5.70 (dd, J=7.1, 3.3 Hz, 1H), 5.49 (dd, J=15.2,2.6 Hz, 1H), 5.30 (ddd, J=18.5, 11.0, 3.2 Hz, 2H), 4.57 (q, J=6.0 Hz,2H), 4.35-4.16 (m, 9H), 4.07-3.95 (m, 2H), 2.17 (s, 3H), 2.08 (s, 3H),2.07 (s, 3H). LRMS (ESI⁻) m/z calcd for C₂₁H₂₉N₅O₁₉P₂ (M−H⁺)=716.09;found 716.3.

Example 3 Synthesis of ca-UDP-2-azido-2-deoxy-D-galactose

Deacetylation of α-UDP-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose,as prepared in example 2, was performed according to Kiso et al.,Glycoconj. J, 2006, 23, 565. Thus,α-UDP-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose (222 mg, 0.309mmol) was dissolved in H₂O (2.5 mL) and Et₃N (2.5 mL) and MeOH (6 mL)were added. The reaction mixture was stirred for 3 h and thenconcentrated in vacuo to afford crude α-UDP-2-azido-2-deoxy-D-galactose.¹H-NMR (300 MHz, D₂O): δ (ppm) 7.99 (d, J=8.2 Hz, 1H), 6.02-5.98 (m,2H), 5.73 (dd, J=7.4, 3.4 Hz, 1H), 4.42-4.37 (m, 2H), 4.30-4.18 (m, 4H),4.14-4.04 (m, 2H), 3.80-3.70 (m, 2H), 3.65-3.58 (m, 1H). LRMS (ESI⁻) m/zcalcd for C₁₅H₂₃N₅O₁₆P₂(M−H⁺)=590.05; found 590.20.

Example 4 Synthesis of α-UDP-D-Galactosamine (UDP-GalNH₂)

To a solution of α-UDP-2-azido-2-deoxy-D-galactose, as prepared inexample 3, in 1:1 H₂O-MeOH mixture (4 mL) was added Lindlar's catalyst(50 mg). The reaction was stirred under a hydrogen atmosphere for 5 hand filtered over celite. The filter was rinsed with H₂O (10 ml) and thefiltrate was concentrated in vacuo to afford α-UDP-D-galactosamine(UDP-GalNH₂) (169 mg, 0.286 mmol, 92% yield over two steps). ¹H-NMR (300MHz, D₂O): δ (ppm) 7.93 (d, J=8.1 Hz, 1H), 5.99-5.90 (m, 2H), 5.76-5.69(m, 1H), 4.39-4.34 (m, 2H), 4.31-4.17 (m, 5H), 4.05-4.01 (m, 1H),3.94-3.86 (m, 1H), 3.82-3.70 (m, 3H), 3.30-3.16 (m, 1H). LRMS (ESI⁻) m/zcalcd for C₁₅H₂₅N₃O₁₆P₂(M−H⁺)=564.06; found 564.10.

Example 5 Synthesis of UDP-GalNProSAc

UDP-α-D-galactosamine (44 mg, 0.078 mmol) was dissolved in 0.1 M NaHCO₃(1 mL). 3-AcS-propionic acid succinimidyl ester (38 mg, 0.156 mmol) andDMF (1 mL) were added and the reaction was stirred overnight at rtfollowed by concentration under reduced pressure. Gradient flashchromatography (7:2:1→5:2:1 EtOAc:MeOH:H₂O) afforded UDP-GalNProSAc (6mg, 0.009 mmol, 11%)+UDP-GalNAc contamination.

LRMS (ESI⁻) calcd for C₂₀H₃₁N₃O₁₈P₂S (M−) 694.08 found 694.1.

¹H-NMR (300 MHz, D₂O): δ (ppm) 7.79 (m, 1H), 5.83-5.80 (m, 2H),5.48-5.45 (m, 1H), 4.28-4.05 (m, 6H), 3.88-3.85 (m, 2H), 3.63-3.55 (m,3H), 3.17-3.16 (m, 2H), 2.60-2.55 (m, 2H) 2.50 (s, 3H).

Example 6 Synthesis of UDP-GalNProSH (11a)

UDP-GalNProSAc (3 mg, 0.005 mmol) was dissolved in degassed 1M NaOH (1mL) and stirred for 1.5 h followed by concentration in vacuo. Theproduct was used crude in the glycosylation experiments.

¹H-NMR (400 MHz, D₂O): δ (ppm) 7.86 (d, J=8.0 Hz, 1H), 5.88-5.86 (m,2H), 5.48-5.45 (m, 1H), 4.20-4.05 (m, 8H), 3.95-3.85 (m, 1H), 3.68-3.65(m, 2H), 2.69-2.67 (m, 2H), 2.60-2.55 (m, 2H).

Example 7 Synthesis of ethyl 2-azido-2, 2-difluoroacetate

To a solution of ethyl 2-bromo-2,2-difluoroacetate (950 mg, 4.68 mmol)in dry DMSO (5 mL) was added sodium azide (365 mg, 5.62 mmol). Afterstirring overnight at room temperature, the reaction mixture was pouredinto water (150 mL). The layers were separated, dichloromethane wasadded to the organic layer and the layer was dried over Na₂SO₄. Afterfiltration, the solvent was removed under reduced pressure (300 mbar) at35° C. affording crude ethyl 2-azido-2,2-difluoroacetate (250 mg, 1.51mmol, 32%).

¹H-NMR (300 MHz, CDCl₃): δ (ppm) 4.41 (q, J=7.2 hz, 2H), 1.38 (t, J=6.9Hz, 3H).

Example 8 Synthesis of α-2-amino-3,4,6-tri-O-acetyl-D-galactose1-phosphate

To a solution ofα-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose-1-phosphate (105 mg,0.255 mmol), as prepared in example 1, in MeOH (3 mL) was added Pd/C (20mg). The reaction was stirred under a hydrogen atmosphere for 2 h andfiltered over celite. The filter was rinsed with MeOH (10 mL) and thefiltrate was concentrated in vacuo to afford the free amine (94 mg,0.244 mmol, 96%).

¹H-NMR (300 MHz, D₂O): δ (ppm) 5.87-5.76 (m, 1H), 5.44 (br. s, 1H),5.30-5.20 (m, 1H), 4.55 (t, J=6.3 Hz, 1H), 4.28-4.00 (m, 3H), 2.11 (s,3H), 2.03 (s, 3H), 2.00 (s, 3H). LRMS (ESI⁻) m/z calcd for C₁₂H₁₉NO₁₁P(M−H⁺)=384.07; found 384.10.

Example 9 Synthesis ofα-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose1-phosphate

To a solution of α-2-amino-3,4,6-tri-O-acetyl-D-galactose 1-phosphate(94 mg, 0.244 mmol), as prepared in example 8, in dry DMF (3 mL), wereadded ethyl 2-azido-2,2-difluoroacetate (48 mg, 0.293 mmol) and Et₃N (68μL, 0.488 mmol). The reaction was stirred for 6 h, followed byconcentration in vacuo to afford the crude product. Gradient flashcolumn chromatography (100:0→50:50 EtOAc:MeOH) affordedα-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose1-phosphate (63 mg, 0.125 mmol, 51%).

Example 10 Synthesis ofUDP-α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose

α-(2′-Azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose1-phosphate, as prepared in example 9, was coupled to UMP according toBaisch et al. Bioorg. Med. Chem., 1997, 5, 383-391, incorporated byreference).

Thus, a solution of D-uridine-5′-monophosphate disodium salt (98 mg,0.266 mmol) in H₂O (1 mL) was treated with DOWEX 50Wx8 (H⁺ form) for 40minutes and filtered. The filtrate was stirred vigorously at rt whiletributylamine (63 μL, 0.266 mmol) was added dropwise. After 30 minutesof further stirring, the reaction mixture was lyophilized and furtherdried over P₂O₅ under vacuum for 5 h. The resulting tributylammoniumuridine-5′-monophosphate was dissolved in dry DMF (15 mL) under an argonatmosphere. Carbonyl diimidazole (35 mg, 0.219 mmol) was added and thereaction mixture was stirred at rt for 30 min. Next, dry MeOH (4.63 μL)was added and stirred for 15 min to remove the excess carbonyldiimidazole. The remaining MeOH was removed under high vacuum (15 min.).Subsequently, N-methylimidazole.HCl (61 mg, 0.52 mmol) was added to thereaction mixture and the resulting compound (63 mg, 0.125 mmol) wasdissolved in dry DMF (15 mL) and added dropwise to the reaction mixture.The reaction was stirred overnight at rt before concentrating themixture under reduced pressure. The consumption of the imidazole-UMPintermediate was monitored by MS analysis. Gradient flash columnchromatography (7:2:1→5:2:1 EtOAc:MeOH:H₂O) affordedUDP-α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose.

¹H-NMR (300 MHz, D₂O): δ (ppm) 7.87 (d, J=8.1 Hz, 1H), 5.913-5.85 (m,2H), 5.67 (dd, J=6.6, 2.7 Hz, 1H), 5.56-5.50 (m, 1H), 5.47-5.43 (m, 1H),5.31-5.25 (m, 2H), 4.61-4.43 (m, 2H), 4.31-4.05 (m, 5H), 2.16 (s, 3H),2.02 (s, 3H), 1.94 (s, 3H).

LRMS (ESI⁻) m/z calcd for C₂₃H₂₉F₂N₆O₂₀P₂(M−H⁺)=809.09; found 809.1.

Example 11 Synthesis ofα-UDP-2-(2′-azido-2′,2′-difluoroacetamido)-2-deoxy-D-galactose(UDP-F2-GalNAz, 11c)

Deacetylation ofUDP-α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactosewas performed according to Kiso et al., Glycoconj. J., 2006, 23, 565,incorporated by reference.

Thus,UDP-α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose,as prepared in example 10, was dissolved in H₂O (1 mL) and triethylamine(1 mL) and MeOH (2.4 mL) were added. The reaction mixture was stirredfor 2 h and then concentrated in vacuo. Gradient flash columnchromatography (7:2:1-5:2:1 EtOAc:MeOH:H₂O) affordedα-UDP-2-(2′-azido-2′,2′-difluoroacetamido)-2-deoxy-D-galactose (11c).

¹H-NMR (300 MHz, D₂O): δ (ppm) 7.86 (d, J=8.1 Hz, 1H), 5.91-5.85 (m,2H), 5.54 (dd, J=6.6, 3.6 Hz, 1H), 4.31-3.95 (m, 9H), 3.74-3.62 (m, 2H).LRMS (ESI⁻) m/z calcd for C₁₇H₂₃F₂N₆O₁₇P₂(M−H⁺)=683.06; found 683.10.

Trimming of Trastuzumab with endoS to Prepare 12

Mass Spectral Analysis of Monoclonal Antibodies

A solution of 50 g (modified) IgG, 1 M Tris-HCl pH 8.0, 1 mM EDTA and 30mM DTT in a total volume of approximately 70 L was incubated for 20minutes at 37° C. to reduce the disulfide bridges allowing to analyzeboth light and heavy chain. If present, azide-functionalities are alsoreduced to amines under these conditions. Reduced samples were washedthree times with milliQ using an Amicon Ultra-0.5, Ultracel-10 Membrane(Millipore) and concentrated to 10 μM (modified) IgG. The reduced IgGwas analyzed by electrospray ionization time-of-flight (ESI-TOF) on aJEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.

Example 12 Preparation of Trimmed Trastuzumab 12

Glycan trimming of trastuzumab was performed with endoS fromStreptococcus pyogenes (commercially available from Genovis, Lund,Sweden). Thus, trastuzumab (10 mg/mL) was incubated with endoS (40 U/mL)in 25 mM Tris pH 8.0 for approximately 16 hours at 37° C. Thedeglycosylated IgG was concentrated and washed with 10 mM MnCl₂ and 25mM Tris-HCl pH 8.0 using an Amicon Ultra-0.5, Ultracel-10 Membrane(Millipore).

A sample was subjected to MS analysis and after deconvolution of peaks,the mass spectrum showed one peak of the light chain and two peaks ofthe heavy chain. The two peaks of heavy chain belonged to one majorproduct (49496 Da, 90% of total heavy chain), resulting from coreGlcNAc(Fuc) substituted trastuzumab, and a minor product (49351 Da, +10%of total heavy chain), resulting from deglycosylated trastuzumab.

Transient Expression of a Trastuzumab Mutant and GlycosyltransferaseEnzymes in CHO

Proteins (enzymes and trastuzumab mutant) were transiently expressed inCHO K1 cells by Evitria (Zurich, Switzerland) at 20-25 mL scale.

GalT double mutant (Y289L, C342T) identified by SEQ ID NO: 1RDLRRLPQLVGVHPPLQGSSHGAAAIGQPSGELRLRGVAPPPPLQNSSKPRSRAPSNLDAYSHPGPGPGPGSNLTSAPVPSTTTRSLTACPEESPLLVGPMLIEFNIPVDLKLVEQQNPKVKLGGRYTPMDCISPHKVAIIIPFRNRQEHLKYWLYYLHPILQRQQLDYGIYVINQAGESMFNRAKLLNVGFKEALKDYDYNCFVFSDVDLIPMNDHNTYRCFSQPRHISVAMDKFGFSLPYVQLFGGVSALSKQQFLSINGFPNNYWGWGGEDDDIYNRLAFRGMSVSRPNAVIGKTRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYMVLEVQRYPLYTKITVDIGTP S

CeGalNAcT [30-383] identified by SEQ ID NO: 2KIPSLYENLTIGSSTLIADVDAMEAVLGNTASTSDDLLDTWNSTFSPISEVNQTSFMEDIRPILFPDNQTLQFCNQTPPHLVGPIRVFLDEPDFKTLEKTYPDTHAGGHGMPKDCVARHRVAIIVPYRDREAHLRIMLHNLHSLLAKQQLDVAIFIVEQVANQTFNRGKLMNVGYDVASRLYPWQCFIFHDVDLLPEDDRNLYTCPIQPRHMSVAIDKFNYKLPYSAIFGGISALTKDHLKKINGFSNDFWGWGGEDDDLATRTSMAGLKVSRYPTQIARYKMIKHSTEATNPVNKCRYKIMGQTKRRWTRDGLSNLKYKLVNLELKPLYTRAVVDLLEKDCRRELRRDFPTCF

CeGalNAcT(30-383)-His₆) identified by SEQ ID NO: 3KIPSLYENLTIGSSTLIADVDAMEAVLGNTASTSDDLLDTWNSTFSPISEVNQTSFMEDIRPILFPDNQTLQFCNQTPPHLVGPIRVFLDEPDFKTLEKTYPDTHAGGHGMPKDCVARHRVAIIVPYRDREAHLRIMLHNLHSLLAKQQLDYAIFIVEQVANQTFNRGKLMNVGYDVASRLYPWQCFIFHDVDLLPEDDRNLYTCPIQPRHMSVAIDKFNYKLPYSAIFGGISALTKDHLKKINGFSNDFWGWGGEDDDLATRTSMAGLKVSRYPTQIARYKMIKHSTEATNPVNKCRYKIMGQTKRRWTRDGLSNLKYKLVNLELKPLYTRAVVDLLEKDCRRELRRDFPTCFHHH HHH

Trastuzumab(heavy chain N300C), identified by SEQ ID NO: 4EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Trastuzumab(light chain), identified by SEQ ID NO: 5DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT KVEIK

Example 13-1 Purification of CeGalNAcT

The purification protocol is based on cation exchange on a SP column (GEHealthcare) followed by size exclusion chromatography.

In a typical purification experiment, CHO-produced supernatantcontaining the expressed CeGalNAcT was dialyzed against 20 mM Trisbuffer, pH 7.5. The supernatant (typically 25 mL) was filtered through a0.45 M-pore diameter filter and subsequently purified over a cationexchange column (SP column, 5 mL, GE Healthcare), which was equilibratedwith 20 mM Tris buffer, pH 7.5 prior to use. Purification was performedon an AKTA Prime chromatography system equipped with an externalfraction collector. Samples were loaded from system pump A. Thenon-bound proteins were eluted from the column by washing the columnwith 10 column volumes (CVs) of 20 mM Tris buffer, pH 7.5. Retainedprotein was eluted with elution buffer (20 mM Tris, 1 NaCl, pH 7.5; 10mL). Collected fractions were analyzed by SDS-PAGE on polyacrylamidegels (12%), and fractions containing the target protein were combinedand concentrated using spin filtration to a volume of 0.5 mL. Next theprotein was purified on a preparative Superdex size exclusion column, onan AKTA purifier system (UNICORN v6.3). This purification step led tothe identification and separation of a dimer, and a monomer fraction oftarget protein. Both fractions were analyzed by SDS-PAGE and stored at-80 OC prior to further use

Example 13-2 Purification of CeGalNAcT-His₆

In a typical purification experiment, CHO supernatant was filteredthrough a 0.45 M-pore diameter filter and applied to a Ni-NTA column (GEHealthcare, 5 mL), which was equilibrated with buffer A (20 mM Trisbuffer, 20 mM imidazole, 500 mM NaCl, pH 7.5) prior to use. Beforefiltration, imidazole was added to the CHO supernatant to a finalconcentration of 20 mM in order to minimize unspecific binding to thecolumn. The column was first washed with buffer A (50 mL). Retainedprotein was eluted with buffer B (20 mM Tris, 500 mM NaCl, 250 mMimidazole, pH 7.5, 10 mL). Fractions were analyzed by SDS-PAGE onpolyacrylamide gels (12%), and the fractions that contained purifiedtarget protein were combined and the buffer was exchanged against 20 mMTris (pH 7.5) by dialysis performed overnight at 4° C. The purifiedprotein was stored at −80° C. prior to further use. Note: for theidentification of the monomeric and dimeric CeGalNAcT-His₆ species anadditional SEC purification was performed (as described above).

Transfer of 11a-c to 12 to Prepare 13a-c

Example 14 Preparation of Trastuzumab(GalNProSH)₂ 13a

Deglycosylated trastuzumab (10 mg/mL) was incubated with UDP-galactosederivative 11a (1.3 mM) and β(1,4)-Gal-T1(Y289L,C342T) (2 mg/mL) in 10mM MnCl₂ and 50 mM Tris-HCl pH 6.0 for 16 hours at 30° C.

Next, the functionalized trastuzumab was incubated with protein Aagarose (40 μL per mg IgG) for 1 hours at rt. The protein A agarose waswashed three times with TBS (pH 6.0) and the IgG was eluted with 100 mMglycine-HCl pH 2.5. The eluted IgG was neutralized with 1 M Tris-HCl pH7.0 and concentrated and washed with 50 mM Tris-HCl pH 6.0 using anAmicon Ultra-0.5, Ultracel-10 Membrane (Millipore) to a concentration of15-20 mg/mL. Spectral analysis after digestion with Fabricator andsubsequent wash with MiliQ using an Amicon Ultra-0.5, Ultracel-10Membrane (Millipore) showed the formation of two products, the majorproduct (24387 Da) with the introduced GalNProSH and the minor (25037Da) with the introduced GalNProSH+UDPGalNProSH as disulfide. The ratiobetween the products is about 60:40.

Glycosyltransfer of a UDP-Galactose Derivative with Gal-TI(Y289L,C342T),General Protocol

Enzymatic introduction of a UDP-galactose derivative onto deglycosylatedtrastuzumab was effected with a the double mutant of bovineβ(1,4)-galactosyltransferase [β(1,4)-Gal-T1(Y289L,C342T)]. Thedeglycosylated trastuzumab (10 mg/mL) was incubated with the appropriateUDP-galactose derivative (0.4 mM) and Gal-T double mutant (1 mg/mL) in10 mM MnCl₂ and 25 mM Tris-HCl pH 8.0 for 16 hours at 30° C. Next, thefunctionalized trastuzumab was incubated with protein A agarose (40 μLper mg IgG) for 2 hours at 4° C. The protein A agarose was washed threetimes with PBS and the IgG was eluted with 100 mM glycine-HCl pH 2.7.The eluted IgG was neutralized with 1 M Tris-HCl pH 8.0 and concentratedand washed with PBS using an Amicon Ultra-0.5, Ultracel-10 Membrane(Millipore) to a concentration of 15-20 mg/mL.

Glycosyltransfer of UDP-GalNAc derivatives with CeGalNAcT (generalprotocol) Enzymatic introduction of GalNAc derivatives onto IgG waseffected with a CeGalNAc-transferase. The deglycosylated IgG (preparedas described above, 10 mg/mL) was incubated with a modified UDP-GalNAcderivative (e.g. an azido-modified sugar-UDP derivative) (0.4 mM) andCeGalNAc-T (1 mg/mL) in 10 mM MnCl₂ and 25 mM Tris-HCl pH 8.0 for 16hours at 30° C. The functionalized IgG (e.g. azido-functionalized IgG)was incubated with protein A agarose (40 μL per mg IgG) for 2 hours at4° C. The protein An agarose was washed three times with PBS and the IgGwas eluted with 100 mM glycine-HCl pH 2.7. The eluted IgG wasneutralized with 1 M Tris-HCl pH 8.0 and concentrated and washed withPBS using an Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore) to aconcentration of 15-20 mg/mL.

Example 15 Preparation of Trastuzumab(GalNAz)₂ 13b with GalT DoubleMutant

Trastuzumab was subjected to the glycosyltransfer protocol withUDP-N-azidoacetylgalactosamine (UDP-GalNAz) and Gal-T double mutant.After protein A affinity purification, mass analysis indicated theformation of a major product (49713 Da, 90% of total heavy chain),resulting from GalNAz transfer to core GlcNAc(Fuc) substitutedtrastuzumab, and a minor product (49566 Da, +10% of total heavy chain),resulting from GalNAz transfer to core GlcNAc substituted trastuzumab.

This is an example of an azido-modified glycoprotein according toformula (13b).

Example 16-1 Preparation of Trastuzumab(GalNAz)₂13b with CeGalNAc-T

Trimmed trastuzumab was subjected to the glycosyltransfer protocol withUDP-N-azidoacetylgalactosamine (UDP-GalNAz) and CeGalNAc-T. Afterprotein A affinity purification, a small sample was reduced with DTT andsubsequently subjected to MS analysis indicating the formation of a onemajor product of (49713 Da, 90% of total heavy chain), resulting fromGalNAz transfer to core GlcNAc(Fuc) substituted trastuzumab, and a minorproduct (49566 Da, +10% of total heavy chain), resulting from GalNAztransfer to core GlcNAc substituted trastuzumab.

Example 16-2 Preparation of Trastuzumab (F₂-GalNAz)₂13c with CeGalNAc-T

Trimmed trastuzumab was subjected to the glycosyltransfer protocol withUDP-N-azidodifluoroacetylgalactosamine (UDP-F₂-GalNAz, 13c) andCeGalNAcT or CeGalNAcT-His₆. After protein A affinity purification asmall sample was reduced with DTT and subsequently subjected to MSanalysis indicating the formation of one major heavy chain product(49865 Da, approximately 90% of total heavy chain), resulting fromF₂-GalNAz transfer to core GlcNAc(Fuc)-substituted trastuzumab which hasreacted with DTT during sample preparation.

This is an example of an azido-modified glycoprotein according toformula (13c).

Synthesis of 17-68

Example 17 Synthesis of 1-Pyrenecarboxylic Acid OSu Ester (45)

To a solution of 1-pyrenecarboxylic acid (65 mg, 0.24 mmol) in DCM/DMF(2 mL each) was added N-hydroxysuccinimide (34 mg, 0.29 mmol) andEDC.HCl (70 mg, 0.36 mmol). The reaction was stirred for 2h andsubsequent diluted with DCM (10 mL), washed with aqueous citric acid(10%, 5 mL) and saturated NaHCO₃ (3×5 mL), dried over Na₂SO₄, filtratedand concentrated in vacuo to give crude 45.

Example 18 Synthesis of (46)

Compound 45 (480 mg, 1.38 mmol) was dissolved in DCM (15 mL) andtert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (348 mg, 1.4 mmoland Et₃N (286 μL, 2.1 mmol) were added. The reaction mixture was stirredovernight and quenched with water (15 mL), the organic layer was washedwith water (1×15 mL) and saturated aqueous NaHCO₃ (2×15 mL), dried overNa₂SO₄, filtrated and concentrated in vacuo. Purification via gradientflash column chromatography (DCM→DCM:MeOH 95:5) yielded the product 46(460 mg, 0.97 mmol, 70%).

Example 19 Synthesis of (17)

Boc-protected pyrene amine 46 (460 mg, 0.97 mmol) was dissolved inmethanol (10 mL) and acetylchloride (140 μL, 1.9 mmol) was added andafter 1 and 3 h additional acetyl chloride (2×140 μL, 1.9 mmol) wasadded. After stirring for 4 h the mixture was concentrated under reducedpressure. Next, the crude product (100 mg, 0.24 mmol) was dissolved inDCM (3 mL) and 2,5-dioxopyrrolidin-1-yl4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate (50 mg, 0.17 mmol) andEt₃N (73 μL, 0.52 mmol) were added. After stirring overnight thesolution was quenched with water (3 mL), washed with water (2×3 mL),dried over Na₂SO₄, filtrated and concentrated. Purification via gradientflash column chromatography (DCM→DCM:MeOH 95:5) yielded the product 17(69 mg, 0.13 mmol, 75%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.48 (d, 1H,J=9.2 Hz), 8.12 (d, 2H, J=7.6 Hz), 8.04-7.93 (m, 6H), 6.59 (bs, 1H),6.38 (s, 2H), 5.99 (bs, 1H), 3.76-3.71 (m, 4H), 3.62-3.60 (m, 2H),3.54-3.52 (m, 2H), 3.37 (t, J=5.2 Hz, 2H), 3.23-3.17 (m, 4H), 1.82 (t,J=6.8 Hz, 2H), 1.63 (q, J=7.2 Hz, 2H).

Example 20-1 Synthesis of Maleimide Alcohol Derivative (47)

To a cooled (0° C.) solution of 5-aminopentan-1-ol (100 mg, 0.97 μmol)in saturated aqueous NaHCO₃ (16 mL) was added N-methoxycarbonylmaleimide(150 mg, 0.64 mol). The mixture was stirred for 1.5 h and extracted withDCM (2×20 mL). The combined organic layers were dried (Na₂SO₄) andconcentrated. The product 47 was obtained as a colorless oil (137 mg,0.75 mmol, 75%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 6.69 (s, 2H), 3.70-3.67(m, 1H), 3.67-3.60 (m, 2H), 3.53 (t, J=7.1 Hz, 2H), 1.68-1.54 (m, 4H),1.42-1.31 (m, 2H).

Example 20-2 Synthesis of Maleimide-Pyrene Derivative (18)

To a solution of alcohol 47 (7.7 mg, 42 μmol) in DCM (1 mL) was addedchlorosulfonyl isocyanate (CSI, 3.9 μL, 5.9 mg, 42 μmol). After thesolution was stirred for 15 min, Et₃N (18 μL, 13 mg, 126 μmol) and asolution of 1-pyrenemethylamine.HCl (49, 11 mg, 42 μmol) and Et₃N (18μL, 13 mg, 126 μmol) were added. After 80 min, saturated aqueous NH₄Cl(20 mL) and DCM (20 mL) were added. After separation, the organic phasewas dried (Na₂SO₄) and concentrated. After gradient flash columnchromatography (DCM→2% MeOH in DCM), product 18 was obtained as aslightly yellow solid (7.4 mg, 14.2 μmol, 34%). ¹H NMR (400 MHz,CDCl₃/CD₃OD) δ (ppm) 8.32-8.26 (m, 1H), 8.20-7.90 (m, 8H), 6.61 (s, 2H),4.90 (s, 2H), 3.65 (t, J=6.5 Hz, 2H), 3.30 (t, J=7.2 Hz, 2H). 1.40-1.20(m, 4H), 1.08-0.97 (m, 2H).

Example 21 Synthesis of BCN-Heptanoic Acid (52)

To a solution of BCN-OSu derivative 51 in MeCN (5 mL) were added7-aminoheptanoic acid 50 (145 mg, 1.0 mmol) in 0.1 M aqueous NaHCO₃ (30mL) and MeCN (25 mL). The mixture was stirred for 4 h and partiallyconcentrated. Aqueous saturated NH₄Cl (30 mL) was added and afterextraction with DCM (2×30 mL), the combined organics were dried (Na₂SO₄)and concentrated. Product 52 was used in the step without furtherpurification. ¹H NMR (400 MHz, CDCl₃) δ (ppm) 4.68 (bs, 1H), 4.14 (d,J=7.9 Hz, 2H), 3.17 (dd, J=12.8, 6.3 Hz, 2H), 2.35 (t, J=7.5 Hz, 2H),2.32-2.09 (m, 6H), 1.70-1.25 (m, 11H), 0.94 (t, J=9.7 Hz, 2H).

Example 22 Synthesis of BCN-Heptanoic Acid Benzylamide (19)

To a mixture of 51 (291 mg, 1.00 mmol) in DCM (25 mL) were added7-aminoheptanoic acid 50 and Et₃N (417 μl, 303 mg, 3.00 mmol) and DMF(10 mL) was added. After evaporation (40° C.) of DCM, the resultingmixture was stirred for 10 min and an aqueous solution (0.1 M) of NaHCO₃was added. After the reaction mixture was stirred for an additional 3 h,it was poured out in saturated aqueous NH₄Cl (50 mL) and extracted withDCM (2×50 mL). The combined organic layers were dried (Na₂SO₄) andconcentrated. The residue was taken up in DCM (25 mL),N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI.HCl,249 mg, 1.30 mmol) and N-hydroxysuccinimide (150, 1.30 mmol) were addedand the resulting mixture was stirred for 18 h. After addition of water(50 mL), the layers were separated and the aqueous phase was extractedwith DCM (2×25 mL). The combined organic layers were washed with brine(50 mL), dried (Na₂SO₄) and concentrated. Column chromatography yieldedthe intermediate NHS ester derivative of 52 as a colorless thick oil(233 mg, 0.56 mmol, 56%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 4.69 (s, 1H),4.14 (d, J=8.0 Hz, 2H), 3.17 (dd, J=13.6, 6.9 Hz, 2H), 2.91-2.77 (m,4H), 2.61 (t, J=7.4 Hz, 2H), 2.37-2.12 (m, 6H), 1.83-1.69 (m, 2H),1.66-1.17 (m, 9H), 1.01-0.90 (m, 2H). Next, to a solution of theintermediate BCN-aminoheptanoic acid NHS ester (49 mg, 0.12 mmol) in DCM(12 mL) were added benzylamine (19 μL, 19 mg, 0.18 mmol) and Et₃N (50μL, 36 mg, 0.36 mmol). The mixture was stirred for 19 h and DCM (10 mL)and saturated aqueous NH₄Cl (20 mL) were added. The organic layer wasdried (Na₂SO₄) and concentrated. After gradient column chromatography(25%-50% EtOAc in heptane) compound 19 was obtained as a white solid (31mg, 0.076 mmol, 63%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.38-7.27 (m, 5H),5.72 (bs, 1H), 4.64 (bs, 1H), 4.45 (d, J=5.6 Hz, 2H), 4.13 (d, J=8.1 Hz,2H), 3.16 (dd, J=12.8, 6.3 Hz, 2H), 2.38-2.13 (m, 8H), 1.75-1.11 (m,11H), 1.00-0.88 (m, 2H).

Example 23 Synthesis of Tert-Butyl N-Benzylsulfamoylcarbamate (53)

Under an atmosphere of N₂, to a cooled solution (−78° C.) oftert-butanol in Et₂O (20 mL) was added chlorosulfonyl isocyanate (CSI)and the mixture was allowed to reach rt. After 45 min, the mixture wasconcentrated and the resulting tert-butyl chlorosulfonylcarbamate wasused in the next step without further purification (considered 68%pure). Thus, to a solution of the crude tert-butylchlorosulfonylcarbamate (199 mg crude=135 mg, 0.63 mmol) in DCM (10 mL)was added Et₃N (263 μL, 191 mg, 1.89 mmol) and benzylamine (82 μL, 81mg, 0.85 mmol). The mixture was stirred for 2 h and quenched withsaturated aqueous NH₄Cl. DCM (10 mL) was added and the layers wereseparated. The organic layer was dried (Na₂SO₄) and concentrated. Aftergradient column chromatography (25%-50% EtOAc in heptane) compound 53was obtained as a white solid (169 mg, 0.59 mmol).

¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.41-7.29 (m, 5H), 5.41-5.30 (m, 1H),4.30-4.20 (m, 2H), 1.46 (s, 9H).

Example 24 Synthesis of N-Benzylsulfamide (54)

To a solution of tert-butyl N-benzylsulfamoylcarbamate 53 (108 mg, 0.38)in DCM (10 mL) was added trifluoroacetic acid (2 mL). The reactionmixture was stirred for 1.5 h and poured into saturated aqueous NaHCO₃(50 mL). After addition of another 50 mL of saturated aqueous NaHCO₃,the aqueous mixture was extracted with DCM (50 mL). The organic layerwas dried (Na₂SO₄) and concentrated. The product 54 was obtained as awhite solid (36 mg, 0.19 mmol, 50%). ¹H NMR (400 MHz, CDCl₃) δ (ppm)7.41-7.30 (m, 5H), 4.32 (d, J=6.1 Hz, 2H).

Example 25 Synthesis of (20)

To a solution of 52 (44 mg, 0.136 mmol) in DCM (5 mL) were addedEDCI.HCl (39 mg, 0.204 mmol), DMAP (2.9 mg, 0.024 mmol) andN-benzylsulfamide 54 (13 mg, 0.068 mmol). After the reaction mixture wasallowed to stir for 22 h at rt, EtOAc (20 mL) and aqueous saturatedNH₄Cl (20 mL) were added. After separation, the aqueous phase wasextracted with EtOAc (20 mL). The combined organic phases were dried(Na₂SO₄) and concentrated. After gradient column chromatography (25%→50%EtOAc in heptane), the product 20 was obtained as an inseparable mixtureof the title compound and N-benzylsulfamide 54 (3.2/1 mass ratio) (14.4mg). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.38-7.27 (m, 5H), 5.99 (bs, 1H),5.01 (t, J=6.2 Hz, 1H), 4.20 (s, 2H), 4.85-4.70 (m, 2H), 4.13 (d, J=8.12Hz, 2H), 3.15 (q, J=6.50, 2H), 2.35-2.15 (m, 6H), 2.09 (t, J=7.4 Hz,2H), 1.65-0.75 (m, 13H).

Example 26 Synthesis of (56)

To a solution of 51 (430 mg, 1.48 mmol) in DCM (20 mL) was added asolution of 5-aminopentan-1-ol 55 (152 mg, 1.47 mmol) in DCM (4 mL) andEt₃N (619 μL, 449 mg, 4.44 mmol). The mixture was stirred for 1.5 h atrt after which a saturated aqueous solution of NaHCO₃ was added (40 mL).After separation, the organic layer was dried (Na₂SO₄) and concentrated.The residue was purified by gradient column chromatography(EtOAc/heptane 1/1→3/1). The product 56 was obtained as a colorlesssticky liquid (356 mg, 1.27 mmol, 81%). ¹H NMR (400 MHz, CDCl₃) δ (ppm)4.68 (s, 1H), 4.14 (d, J=8.0 Hz, 2H), 3.65 (dd, J=11.7, 6.3 Hz, 2H),3.19 (dd, J=13.2, 6.7 Hz, 2H), 2.35-2.15 (m, 6H), 1.66-1.30 (m, 7H),1.02-0.88 (m, 2H).

Example 27 Synthesis of (21)

To a solution of 56 (51 mg, 0.18 mmol) in DCM (10 mL) was addedchlorosulfonyl isocyanate (16 μl, 25 mg, 0.18 mmol). After the mixturewas stirred for 40 min, Et₃N (75 μl, 55 mg, 0.54 mmol) and benzylamine(19 μl, 19 mg, 0.18 mmol) were added.

The mixture was stirred for an additional 1.5 h and quenched throughaddition of an aqueous solution of NH₄Cl (sat). After separation, theaqueous layer was extracted with DCM (20 mL). The combined organiclayers were dried (Na₂SO₄) and concentrated. The residue was purified bygradient column chromatography (20%→50% EtOAc in pentane) and product 21was obtained as colorless thick oil (57 mg, 0.12 mmol, 67%). ¹H NMR (400MHz, CDCl₃) δ (ppm) 7.41-7.28 (m, 5H), 5.55 (s, 1H), 4.75 (s, 1H),4.29-4.24 (m, 2H), 4.20-4.08 (m, 2H), 3.19 (dd, J=13.4, 6.6 Hz, 2H),2.37-2.16 (m, 6H), 1.74-1.31 (m, 9H), 0.94 (t, J=9.7 Hz, 2H).

Example 28 Synthesis of (22)

Under an inert atmosphere, 21 (93 mg, 0.19 mmol) was dissolved inanhydrous THF (10 mL). PPh₃ (49 mg, 0.19 mmol) and MeOH (50 μL, 1.23mmol) were added and the mixture was cooled to 0° C. A solution of DIAD(37 μL, 0.19 mmol) in anhydrous THF (5 mL) was slowly added and themixture was allowed to reach rt, after which the reaction was stirredfor 18 h and subsequently concentrated. Gradient column chromatography(20→50% EtOAc in heptane) yielded product 22 as colorless thick oil. ¹HNMR (400 MHz, CDCl₃) δ (ppm) 7.39-7.26 (m, 5H), 5.94-5.84 (m, 1H),4.80-4.64 (m, 1H), 4.19 (d, J=6.4 Hz, 2H,), 4.13 (d, J=7.4 Hz, 2H), 4.08(t, J=6.5 Hz, 2H), 3.17 (q, 2H, J=6.5 Hz), 3.12 (s, 3H), 2.35-2.14 (m,6H), 1.80-1.65 (m, 13H).

Example 29 Synthesis of (58)

To a solution of 57 (1.5 g, 10 mmol) in DCM (150 mL), under a N₂atmosphere, was added CSI (0.87 mL, 1.4 g, 10 mmol), Et₃N (2.8 mL, 2.0g, 20 mmol) and 2-(2-aminoethoxy)ethanol (1.2 mL, 1.26 g, 12 mmol). Themixture was stirred for 10 min and quenched through addition of aqueousNH₄Cl (sat., 150 mL). After separation, the aqueous layers was extractedwith DCM (150 mL). The combined organic layers were dried (Na₂SO₄) andconcentrated. The residue was purified with column chromatography.Product 58 was obtained as slightly yellow thick oil (2.06 g, 5.72 mmol,57%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 6.0 (bs, 1H), 4.28 (d, J=8.2 Hz,2H), 3.78-3.73 (m, 2H), 3.66-3.61 (m, 2H), 3.61-3.55 (m, 2H), 3.34 (t,J=4.9 Hz, 2H), 2.37-2.15 (m, 6H), 1.64-1.48 (m, 2H), 1.40 (quintet,J=8.7 Hz, 1H), 1.05-0.92 (m, 2H).

Example 30 Synthesis of (59)

To a solution of 58 (130 mg, 0.36 mmol) were subsequently added CSI (31μL, 51 mg, 0.36 mmol), Et₃N (151 μL) and 2-(2-aminoethoxy)ethanol (36μL, 38 mg, 0.36 mmol). After 15 min, water (20 mL) was added and afterseparation, the aqueous layer was acidified with 1 M aq. HCl to pH 3 andextracted with DCM (20 mL). The DCM layer was dried and concentrated.After column chromatography, the product 59 was obtained as colorlessoil (87 mg, 0.15 mmol, 42%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 6.15-5.95(m, 2H), 4.40-4.32 (m, 2H), 4.31 (d, J=8.3 Hz, 2H), 3.85-3.55 (m, 10H),3.45-3.25 (m, 4H), 2.40-2.15 (m, 6H), 1.65-1.47 (m, 2H), 1.40 (quintet,J=8.7 Hz, 1H), 1.06-1.92 (m, 2H).

Example 31 Synthesis of (23)

To a solution of 59 (63 mg, 0.11 mmol) in DCM (10 mL) were subsequentlyadded p-nitrophenyl chloroformate (22 mg, 0.11 mmol) and Et₃N (46 μL, 33mg, 0.33 mmol). After 20 h, benzylamine (22 μL, 21.6 mg, 0.20 mmol) wasadded to the reaction mixture. The mixture was stirred for an additional24 h where after the mixture was concentrated and the residue waspurified by gradient column chromatography (1st col. 0-20% MeOH in DCM,2nd col. 0-8% MeOH in DCM). Product 23 was obtained as a colorless film(18 mg, 0.026 mmol, 23%). ¹H NMR (400 MHz, CD₃OD) δ (ppm) 7.38-7.18 (m,5H), 4.31-4.22 (m, 6H), 4.22-4.16 (m, 2H), 3.70-3.63 (m, 4H), 3.63-3.54(m, 4H), 3.34 (s, 1H), 3.24-3.15 (m, 4H), 2.30-2.10 (m, 6H), 1.68-1.52(m, 2H), 1.42 (quintet, J=8.7 Hz, 1H), 1.02-0.90 (m, 2H).

Example 32 Synthesis of BCN-dPEG₄-C(O)OSu (60a)

To a solution of amino-dPEG₄-acid (1.23 g, 4.23 mmol) in anhydrous DMF(30 mL) were subsequently added 51 (1.02 g, 3.85 mmol) and triethylamine(1.60 mL, 11.53 mmol). The reaction mixture was stirred for 3h at rt,after which EDCI.HCl (0.884 g, 4.61 mmol) and NHS (88 mg, 0.77 mmol)were added. The resulting solution was stirred overnight at rt andpoured into 100 mL NaHCO₃ (sat.) and 150 mL EtOAc. The layers wereseparated and the organic phase was washed with sat. NaHCO₃ (90 mL) andH₂O (75 mL). The organic phase was dried (Na₂SO₄), filtered andconcentrated in vacuo. Gradient flash chromatography (MeCN→MeCN:H₂O30:1) afforded product 60a as colorless oil (800 mg, 1.48 mmol, 40%).

Example 33 Synthesis of BCN-dPEG₄-Pyrene (24)

To a solution of 60a (50 mg, 0.095 mmol) in DCM (10 mL) was added 49 (30mg, 0.11 mmol) and Et₃N (17 μL, 0.12 mmol). After stirring overnight atrt, the reaction mixture was concentrated under reduced pressure.Subsequent purification via flash column chromatography (DCM→DCM:MeOH9:1) yielded product 24 (38 mg, 61%). ¹H NMR (400 MHz, CDCl₃) δ (ppm)8.29-8.26 (m, 2H), 8.19-8.11 (m, 4H), 8.06-7.97 (m, 4H), 7.04 (br. s,1H), 5.24 (br. s, 1H), 5.16 (d, 2H, J=4 Hz), 4.06 (d, 2H, J=4 Hz), 3.76(t, 2H, J=5.6 Hz), 3.50 (m, 2H), 3.39-3.22 (m, 14H), 2.56-2.53 (m, 2H),2.27-2.13 (m, 7H), 1.29-1.25 (m, 2H), 0.87-0.83 (m, 2H).

Example 34 Synthesis of BCN-PEG₈-C(O)OSu (60b)

To a solution of amino-dPEG₈-acid (217 mg, 0.492 mmol) in anhydrous DMF(3 mE) were subsequently added 51 (143 mg, 0.492 mmol) and Et₃N (204 μL,1.47 mmol). The reaction mixture stirred for 3h at rt, after whichEDCI.HCl (0.88 g, 4.61 mmol) and NHS (88 mg, 0.77 mmol) were added. Theresulting solution was stirred overnight at rt and poured into 50 mLNaHCO₃ (sat.) and 50 mL EtOAc. The layers were separated and the organicphase was washed with sat. NaHCO₃ (50 mL) and H₂O (30 mL). The organicphase was dried (Na₂SO₄), filtered and concentrated in vacuo. Gradientflash chromatography (MeCN→MeCN:H₂O 30:1) afforded product 60b ascolorless oil (212 mg, 0.30 mmol, 60%).

¹H NMR (300 MHz, CDCl₃): δ (ppm) 4.13 (d, J=8.1 Hz, 2H), 3.84 (t, J=6.3Hz, 2H), 3.68-3.59 (m, 28H), 3.54 (t, J=5.1 Hz, 2H), 3.36 (q, J=5.4 Hz,2H), 2.89 (t, J=6.3 Hz, 2H), 2.82 (s, 4H), 2.35-2.15 (m, 6H), 1.68-1.48(m, 2H), 1.44-1.23 (m, 1H), 1.00-0.86 (m, 2H). LRMS (ESI+) m/z calcd forC₃₄H₅₄N₂O₁₄ (M+Na⁺)=737.8; found 737.3.

Example 35 Synthesis of BCN-PEG₈-Pyrene (25)

To a solution of 60b (100 mg, 0.14 mmol) in DCM (15 mL) was added1-aminomethylpyrene.HCl 49 (50 mg, 0.19 mmol) and Et₃N (47 μL, 0.25mmol). After stirring for 3 h, the reaction mixture was concentratedunder reduced pressure. Subsequent purification via flash columnchromatography (DCM→DCM:MeOH 95:5) yielded the product 25 (35 mg, 30%).¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.30-8.28 (m, 1H), 8.21-8.13 (m, 4H),8.05-7.99 (m, 4H), 7.22 (bs, 1H), 5.16 (d, 2H, J=5.2 Hz), 4.12 (d, 2H,J=8.0 Hz), 3.76 (t, 2H, J=6.0 Hz), 3.65-3.49 (m, 21H), 3.45-3.42 (m,2H), 3.36-3.32 (m, 4H), 2.59-2.54 (m, 4H), 2.28-2.20 (m, 4H), 1.59-1.54(m, 2H), 1.38-1.25 (m, 2H), 0.93-0.91 (m, 2H).

Example 36 Synthesis of (61)

To a solution of 57 (0.15 g, 1.0 mmol) in DCM (15 mL) was added CSI (87μL, 0.14 g, 1.0 mmol), Et₃N (279 μL, 202 mg, 2.0 mmol) and a solution ofH₂N-PEG₃-OH (251 mg, 1.3 mmol) in DCM (1 mL). After stirring for 2.5 h,the reaction mixture was quenched through addition of a solution ofNH₄Cl (sat., 20 mL). After separation, the aqueous layer was extractedwith DCM (20 mL). The combined organic layers were dried (Na₂SO₄) andconcentrated. The residue was purified by gradient column chromatography(0-10% MeOH in DCM). Product 61 was obtained as colorless thick oil (254mg, 0.57 mmol, 57%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 6.81 (br. s, 1H),4.26 (d, J=8.2 Hz, 2H), 3.80-3.70 (m, 4H), 3.70-3.58 (m, 10H), 3.36 (t,J=4.7 Hz, 2H), 2.36-2.16 (m, 6H), 1.64-1.49 (m, 2H), 1.40 (quintet,J=8.7 Hz, 1H), 1.04-0.92 (m, 2H).

Example 37 Synthesis of (62)

To a solution of 61 (242 mg, 0.54 mmol) in DCM (30 mL) were addedp-nitrophenyl chloroformate (218 mg; 1.08 mmol) and Et₃N (226 μL, 164mg, 1.62 mmol). The mixture was stirred for 17 h. and quenched withwater (20 mL). After separation, the organic layer was dried (Na₂SO₄)and concentrated. The residue was purified by gradient columnchromatography (EtOAc/pentane 1/1→EtOAc) to afford product 62 (259 mg,0.38 mmol). LRMS (ESI) m/z calcd for C₃₃H₄₂N₅O₁₆S (M+NH₄₊)=796.23; found796.52.

Example 38 Synthesis of BCN-Sulfamide-Pyrene (26)

Compound 62 (40 mg, 0.11 mmol) was dissolved in DCM (10 mL) and 49 (34mg, 0.13 mmol) and Et₃N (30 μL, 0.21 mmol) were added. The reactionmixture was stirred for 4 h, concentrated under reduced pressure andpurified on gradient flash column chromatography (DCM→DCM:MeOH 96:4).The fractions containing the product were washed with sat. NaHCO₃ (3×100mL), dried over Na₂SO₄, filtrated and concentrated in vacuo to yield 26(25 mg, 36%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.29-8.26 (m, 2H),8.19-8.11 (m, 4H), 8.06-7.97 (m, 4H), 6.87 (d, 1H, J=12 Hz), 5.78 (br.s, 2H), 5.12 (d, 2H, J=5.6 Hz), 4.31-4.26 (m, 2H), 3.97 (d, 2H, J=8.4Hz), 3.69-3.62 (m, 4H), 3.28 (m, 2H), 2.18-1.96 (m, 7H), 1.28 (m, 1H),0.79-0.74 (m, 2H).

Example 39 Synthesis of DIBAC-PEG₄-Pyrene (27)

Compound 45 (75 mg, 0.22 mmol) was dissolved in DCM (3 mL) followed bythe addition of amino-PEG₄-carboxylic acid (53 mg, 0.20 mmol) and Et₃N(83 μL, 0.6 mmol). After stirring overnight additional 45 (25 mg, 0.07mmol) was added and the reaction mixture was stirred for an additional 1h. After complete conversion (based on TLC-analysis),N-hydroxysuccinimide (5 mg, 0.04 mmol) and EDC.HCl (70 mg, 0.36 mmol)were added and the reaction was stirred overnight at rt. To the reactionmixture was added 64 (60 mg, 0.2 mmol) and Et₃N (50 μL, 0.36 mmol) wereadded and after 1 h the reaction mixture was concentrated under reducedpressure. Purification via gradient flash column chromatography(DCM→DCM:MeOH 9:1) yielded product 27 (12 mg, 8%). ¹H NMR (400 MHz,CDCl₃) δ (ppm) 8.61 (d, 1H, J=9.2 Hz), 8.23-8.21 (m, 2H), 8.17-8.10 (m,4H), 8.07-8.02 (m, 3H), 7.59 (d, 1H, J=7.2 Hz), 7.38-7.18 (m, 7H), 7.06(bs, 1H), 6.45 (m, 1H), 5.00 (d, 1H, J=13.6), 3.86-3.79 (m, 3H),3.70-3.19 (m, 14H), 2.42-2.38 (m, 1H), 2.19-2.15 (m, 2H), 1.89-1.85 (m,1H).

Example 40 Synthesis of DIBAC-PEG-Pyrene (28)

Compound 45 (24 mg, 0.07 mmol) was dissolved in DCM (1 mL) followed bythe addition of amino-PEGs-carboxylic acid (27 mg, 0.06 mmol) and Et₃N(14 μL, 0.10 mmol). After stirring for 2 h, additional 45 (10 mg, 0.03mmol) was added and the reaction mixture was stirred for on.Subsequently, NHS (10 mg, 0.08 mmol) and EDC.HCl (17 mg, 0.09 mmol) wereadded and the reaction was stirred for 2h. Next 64 (21 mg, 0.08 mmol)and Et₃N (14 μL, 0.10 mmol) were added and after 4 h the reaction wasquenched by the addition of water (3 mL). The organic layer was washedwith water (2×3 mL), dried over Na₂SO₄, filtrated and concentrated underreduced pressure. Purification via flash column chromatography(DCM→DCM:MeOH 93:7) yielded the product (8 mg, 14%). ¹H NMR (400 MHz,CDCl₃) δ (ppm) 8.55 (d, 1H, J=9.2 Hz), 8.19-7.86 (m, 7H), 7.59 (d, 1H,J=6.8 Hz), 7.33-7.18 (m, 8H), 6.99 (m, 1H), 6.45 (m, 1H), 5.03 (d, 1H,J=14.0), 3.78-3.74 (m, 3H), 3.65-3.29 (m, 29H), 2.43-2.37 (m, 1H)2.25-2.21 (m, 2H), 1.91-1.84 (m, 1H).

Example 41 Synthesis of (65)

Compound 45 (50 mg, 0.14 mmol) was dissolved in DCM (3 mL) followed bythe addition of 2-aminoethanol (10 μL, 0.16 mmol) and Et₃N (30 μL, 0.22mmol). After 2 h, additional 2-aminoethanol (10 μL, 0.16 mmol) was addedand the reaction mixture was stirred overnight at rt. Subsequently,water (5 mL) was added and the organic layer was washed with water (3×5mL), dried over Na₂SO₄, filtrated and concentrated in vacuo.Purification via gradient flash column chromatography (DCM→DCM:MeOH97:3) yielded product 65 (32 mg, 79%). LRMS (ESI⁺) m/z calcd forC₁₉H₁₆NO₂ (M+H⁺)=290.12; found 290.30.

Example 42 Synthesis of DIBAC-Sulfamide-Pyrene (29)

Compound 65 (26 mg, 0.09 mmol) was dissolved in DCM (2 mL) followed bythe addition of CSI (8 μL, 0.09 mmol). After 5 min. Et₃N (37 μL, 0.27mmol) and 64 (26 mg, 0.09 mmol) were added and the reaction was stirredfor 2 h at rt, after which the reaction was quenched through theaddition of aqueous NH₄Cl (sat., 5 mL). The organic layer was washedwith water (3×5 mL), dried over Na₂SO₄, filtrated and concentrated invacuo. Purification via gradient flash column chromatography(DCM→DCM:MeOH 95:5) yielded product 29 (10 mg, 17%). ¹H NMR (400 MHz,CDCl₃) δ (ppm) 8.49 (d, 1H, J=9.2 Hz), 8.25-8.18 (m, 3H), 8.11-7.95 (m,5H), 7.38-7.00 (m, 8H), 6.05 (m, 1H), 5.96 (m, 1H), 4.62-4.58 (m, 1H).4.52-4.47 (m, 1H), 4.11-4.08 (m, 1H), 3.81-3.78 (m, 1H), 3.64-3.60 (m,1H), 3.06 (d, 1H, J=14 Hz), 2.90-2.87 (m, 2H), 2.17-2.09 (m, 1H),1.62-1.56 (m, 2H).

Example 43-1 Synthesis of (30)

A solution of BCN-PEG₄-C(O)OSu (60a, 7.1 mg, 0.013 mmol) and Et₃N (9.1μL, 6.6 mg, 65.5 μmol) in 1 mL DMF was added to H-Ahx-maytansin.TFA (10mg, 0.011 mmol). The reaction was stirred for 20 h at rt andsubsequently concentrated under reduced pressure. The residue waspurified via reversed phase (C18) HPLC chromatography (30-90% MeCN (1%AcOH) in H₂O (1% AcOH). Product 30 was obtained as colorless liquid (8.9mg, 7.5 μmol, 68%). LRMS (ESI⁺) m/z calcd for C₆₀H₈₇ClN₅O₁₆(M⁺−H₂O)=1168.58; found 1168.87.

Example 43-2 Synthesis of BCN-PEG₁₂-C(O)OSu

To a solution of amino-dPEG₁₂-acid (43 mg, 0.069 mmol) in anhydrous DMF(1 mL) were added 51 (22 mg, 0.076 mmol) and triethylamine 24 μL, 0.174mmol). The reaction mixture stirred for 5 h at rt, after which EDCI.HCl(27 mg, 0.139 mmol) and NHS (8 mg, 0.076 mmol) were added. The resultingsolution was stirred overnight at rt and poured into 10 mL sat. NaHCO₃and 10 mL DCM. The layers were separated and the organic phase waswashed with H₂O (2×10 mL). The combined water layers were extracted withDCM (10 mL). The combined organic layers were dried (Na₂SO₄), filteredand concentrated in vacuo. Gradient flash column chromatography (MeCN-MeCN:H₂O 20:1, 10:1) afforded BCN-PEG₁₂-C(O)OSu.

¹H NMR (400 MHz, CDCl₃): δ (ppm) 4.14 (d, J=8.0 Hz, 2H), 3.85 (t, J=6.4Hz, 2H), 3.69-3.60 (m, 44H), 3.56 (t, J=5.2 Hz, 2H), 3.36 (q, J=5.2 Hz,2H), 2.91 (t, J=6.4 Hz, 2H), 2.84 (s, 4H), 2.36-2.17 (m, 6H), 1.65-1.51(m, 2H), 1.44-1.23 (, J=8.4 Hz, 1H), 1.00-0.88 (m, 2H). LRMS (ESI+) m/zcalcd for C₄₂H₇₀N₂O₁₈ (M+H⁺)=892.0; found 891.6.

Example 44 Synthesis of (31)

A solution of BCN-PEG₁₂-C(O)OSu (14 mg, 15.7 μmol) and Et₃N (9.1 μL, 6.6mg, 65.5 μmol) in 1.1 mL DMF was added to H-Ahx-maytansin.TFA (10 mg,0.011 mmol). After 18 h, 2,2′-(ethylenedioxy)bis(ethylamine) (2.3 μL,2.3 mg, 16 μmol) was added and the mixture was concentrated. The residuewas purified via reversed phase (C18) HPLC chromatography (30-90% MeCN(1% AcOH) in H₂O (1% AcOH). Product 31 was obtained as a colorless film(6.6 mg, 4.3 μmol, 39%). LRMS (ESI⁺) m/z calcd for C₇₅H₁₁₈ClN₅O₂₅(M⁺−H₂O)=1520.79; found 1520.96.

Example 45 Synthesis of BCN-PEG₂₄-C(O)OSu

To a solution of amino-dPEG₂₄-acid (48 mg, 0.042 mmol) in anhydrous DMF(1 mL) were added 51 (14 mg, 0.048 mmol) and triethylamine (17 μL, 0.125mmol). The reaction mixture was stirred at rt overnight, after which DCM(10 mL) and citric acid (10% aq. sol., 5 mL) were added. The water phasewas extracted with DCM (15 mL) and the combined organic layers weredried (Na₂SO₄). After filtration the solvent was removed in vacuo. Thecrude product was redissolved in anhydrous DMF (1 mL) and EDCI.HCl (17mg, 0.088 mmol) and NHS (8 mg, 0.070 mmol) were added. The resultingsolution was stirred overnight at rt, after which an addition equivalentof EDCI.HCL (16 mg) and NHS (7 mg) were added. After 6h the reactionmixture was poured into 10 mL sat. NaHCO₃ and 15 mL EtOAc. The layerswere separated and the organic phase was washed with sat. NaHCO₃ (10 mL)and H₂O (10 mL). The organic phase was dried (Na₂SO₄), filtered andconcentrated in vacuo. Gradient flash column chromatography(MeCN→MeCN:H₂O 20:1, 10:1, 5:1) afforded BCN-PEG₂₄-C(O)OSu as acolorless oil (32 mg, 0.022 mmol, 54%).

¹H NMR (400 MHz, CDCl₃): δ (ppm) 4.08 (d, J=8.0 Hz, 2H), 3.78 (t, J=6.4Hz, 2H), 3.63-3.54 (m, 92H), 3.49 (t, J=5.2 Hz, 2H), 3.30 (q, J=5.2 Hz,2H), 2.84 (t, J=6.4 Hz, 2H), 2.78 (s, 4H), 2.29-2.08 (m, 6H), 1.59-1.43(m, 2H), 1.35-1.23 (m, 1H), 0.93-0.80 (m, 2H). LRMS (ESI+) m/z calcd forC₆₆H₁₁₈N₂O₃₀ (M+H⁺)=1420.66; found 1420.0.

Example 46 Synthesis of (32)

A solution of BCN-PEG₂₄-C(O)OSu (25 mg, 17.6 μmol) and Et₃N (9.1 μL, 6.6mg, 65.5 μmol) in 0.78 mL DMF was added to H-Ahx-maytansin.TFA (10 mg,0.011 mmol). After 18 h, 2,2′-(ethylenedioxy)bis(ethylamine) (2.3 μL,2.3 mg, 16 μmol) was added and the mixture was concentrated. The residuewas purified via reversed phase (C18) HPLC chromatography (30-90% MeCN(1% AcOH) in H₂O (1% AcOH). The product was obtained as a colorless film(11.4 mg, 5.5 μmol, 50%). LRMS (ESI⁺) m/z calcd for C₁₀₀H₁₇₁ClN₅O₃₇ ³⁺(M+3H⁺)/3=690.05; found 690.05.

Example 47 Synthesis of (33)

A solution of H-Ahx-maytansin.TFA (20 mg, 0.023 mmol) in DMF (2 mL) wasadded to a solution of 62 (14 mg, 0.023 mmol) and Et₃N (9.5 μL, 6.9 mg,0.068 mmol) in DMF (2 mL) and the resulting reaction mixture was stirredfor 24 h. Title compound 33 was obtained in quantitative yield aftersilica gel column chromatography (29 mg, +99%). Proof of identity wasperformed at ADC stage.

Example 48 Synthesis of (34)

A solution of 60a (6.6 mg, 0.012 mmol) and Et₃N (6.8 μL, 4.9 mg, 48.5μmol) in 1 mL DMF was added to H-Val-Cit-PABA-duocarmycin (10 mg, 0.0097mmol). After 18 h, 2,2′-(ethylenedioxy)bis(ethylamine) (1.8 μL, 1.8 mg,12 μmol) was added and the mixture was concentrated under reducedpressure. The residue was purified via reversed phase (C18) HPLCchromatography (30-90% MeCN (1% AcOH) in H₂O (1% AcOH). Product 34 wasobtained as a colorless film (7.5 mg, 5.1 μmol, 53%).

LRMS (ESI⁺) m/z calcd for C₇₁H₉₄ClN₁₀O₂₁ (M+H⁺)=1457.63; found 1456.89.

Example 49 Synthesis of (35)

A solution of 62 (6.0 mg, 9.8 μmol) and Et₃N (6.8 μL, 4.9 mg, 48.5 μmol)in DMF (1 mL) was added to H-Val-Cit-PABA-duocarmycin (10 mg, 0.0097mmol). After 22 h, 2,2′-(ethylenedioxy)bis(ethylamine) (2.8 μL, 2.8 mg,19 μmol) was added. After 1h, the reaction mixture was concentratedunder reduced pressure and the residue was purified via reversed phase(C18) HPLC chromatography (30-90% MeCN (1% AcOH) in H₂O (1% AcOH).Product 35 was obtained as a white solid (6.4 mg, 4.2 μmol. 44%). LRMS(ESI⁺) m/z calcd for C₆₉H₉₂ClN₁₂O₂₂S (M+H⁺)=1507.59; found 1508.00.

Example 50 Synthesis of (36)

To a solution of 58 (229 mg, 0.64 mmol) in DCM (20 mL) were addedp-nitrophenyl chloroformate (128 mg, 0.64 mmol) and Et₃N (268 μL, 194mg, 1.92 mmol). The mixture was stirred overnight at rt and subsequentlyconcentrated under reduced pressure. The residue was purified viagradient column chromatography (20-70% EtOAc in heptane (1% AcOH) toafford the PNP carbonate derivative of 58 as a white solid (206 mg, 0.39mmol, 61%). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.31-8.26 (m, 2H), 7.45-7.40(m, 2H), 5.56 (t, J=6.0 Hz, 1H), 4.48-4.40 (m, 2H), 4.27 (d, J=8.2 Hz,2H), 3.81-3.75 (m, 2H), 3.68 (t, J=5.0 Hz, 2H), 3.38-3.30 (m, 2H),2.36-2.14 (m, 6H), 1.61-1.45 (m, 2H), 1.38 (quintet, J=8.7 Hz, 1H),1.04-0.94 (m, 2H).

Next, to a solution of the PNP-derivative of 58 (4.1 mg, 7.8 μmol) andEt₃N (3.3 μL, 2.4 mg, 23.4 μmol) in DMF (1 mL) was added a solution ofH-Val-Cit-PABA-Ahx-maytansin (10 mg, 8.6 μmol) in DMF (100 μL). After 20h, 2,2′-(ethylenedioxy)bis(ethylamine) (5.7 μL, 5.6 mg, 38 μmol) wasadded and the mixture was concentrated under reduced pressure. Theresidue was purified via reversed phase (C18) HPLC chromatography(30-90% MeCN (1% AcOH) in H₂O (1% AcOH) to give 36 (2.2 mg, 1.4 μmol,18%). LRMS (ESI⁺) m/z calcd for C₇₃H₁₀₃ClN₁₁O₂₁S (M−18+H⁺)=1536.67;found 1537.08.

Example 51 Synthesis of (66)

To a stirring solution of 57 (500 mg, 3.33 mmol) in DCM (100 mL) wasadded CSI (290 μL, 471 mg, 3.33 mmol). After 20 min, Et₃N (1.4 mL, 1.0g, 10 mmol) and a solution of diethanolamine.HCl (571 mg, 4.0 mmol) inDMF (5 mL) were added subsequently. After an additional 45 min., thereaction mixture was concentrated under reduced pressure and the residuewas purified by gradient column chromatography (0-15% MeOH in DCM).Product 66 was obtained as colorless thick oil (767 mg, 2.13 mmol, 64%).¹H NMR (400 MHz, CDCl₃) δ (ppm) 4.26 (d, J=8.2 Hz, 2H), 3.87 (t, J=4.9Hz, 4H), 3.55 (t, J=4.9 Hz, 4H), 2.37-2.16 (m, 6H), 1.65-1.45 (m, 2H),1.39 (quintet, J=8.6 Hz, 1H), 1.05-0.92 (m, 2H)

Example 52 Synthesis of (67)

To a suspension of 66 (206 mg, 0.57 mmol) in DCM (20 mL) was added Et₃N(318 μL, 231 mg, 2.28 mmol) and p-nitrophenyl chloroformate (230 mg,1.14 mmoL, 2 eq.). The reaction mixture was stirred for 28 h at rt andsubsequently concentrated. The residue was purified via gradient columnchromatography (20-75% EtOAc in heptane), yielding 67 as slightly yellowthick oil (83 mg, 0.12 mmol, 21%). ¹H NMR (400 MHz, CDCl₃) δ (ppm)8.31-8.24 (m, 4H), 7.43-7.34 (m, 4H), 4.53 (t, J=5.4 Hz, 2H), 4.22 (d,J=4.2 Hz, 2H), 3.87 (t, J=5.4 Hz, 4H), 2.35-2.15 (m, 6H), 1.55-1.40 (m,2H), 1.35 (quintet, J=8.8 Hz, 1H), 1.03-0.92 (m, 2H).

Example 53 Synthesis of (37)

A solution of 67 (2.7 mg, 3.9 μmol) and Et₃N (2.7 μL, 2.0 mg, 19.5 μmol)in DMF (1 mL) was added to a solution of H-Val-Cit-PABA-Ahx-maytansin(10 mg, 0.0086 mmol) in DMF (100 μL). The mixture was allowed to reactovernight at rt, and subsequently concentrated. A solution of2,2′-(ethylenedioxy)bis(ethylamine) (2.8 μL, 2.8 mg, 19 μmol) in DMF (1mL) was added. The reaction mixture was concentrated and the residue waspurified via reversed phase (C18) HPLC chromatography (30-90% MeCN (1%AcOH) in H₂O (1% AcOH) to give compound 37 (4.8 mg, 1.7 μmol, 45%). LRMS(ESI⁺) m/z calcd for C₁₃₁H₁₈₆Cl₂N₂₀O₃₈S (M+2H⁺)/2=1375.12; found1375.51.

Example 54 Synthesis of (68)

To a stirring solution of 58 (47 mg, 0.13 mmol) in DCM (10 mL) was addedCSI (11 μL, 18 mg, 0.13 mmol). After 30 min, Et₃N (91 μL, 66 mg, 0.65mmol) and a solution of diethanolamine (16 mg, 0.16 mmol) in DMF (0.5mL) were added. After 30 minutes p-nitrophenyl chloroformate (52 mg,0.26 mmol) and Et₃N (54 μL, 39 mg, 0.39 mmol) were added. After anadditional 4.5 h, the reaction mixture was concentrated and the residuewas purified by gradient column chromatography (33-66% EtOAc/heptane (1%AcOH)) to afford 68 as colorless oil (88 mg, 0.098 mmol, 75%). ¹H NMR(400 MHz, CDCl₃) δ (ppm) 8.28-8.23 (m, 4H), 7.42-7.35 (m, 4H), 4.52 (t,J=5.4 Hz, 4H), 4.30 (d, J=8.3 Hz, 2H), 4.27-4.22 (m, 2H), 3.86 (t, J=5.3Hz, 4H), 3.69-3.65 (m, 2H), 3.64-3.59 (m, 2H), 3.30-3.22 (m, 2H),2.34-2.14 (m, 6H), 1.62-1.46 (m, 2H), 1.38 (quintet, J=8.7 Hz, 1H),1.04-0.92 (m, 2H).

Example 55 Synthesis of (38)

A solution of 68 (3.9 mg, 4.3 μmol) and Et₃N (3.0 μL, 2.2 mg, 21.5 μmol)in DMF (1 mL) was added to a solution of H-Val-Cit-PABA-Ahx-maytansin(10 mg, 8.6 μmol) in DMF (100 μL). The mixture was allowed to react o.n.and concentrated. The residue was purified via reversed phase (C18) HPLCchromatography (30-90% MeCN (1% AcOH) in H₂O (1% AcOH) to give product38 (3.9 mg, 1.32 μmol, 31%). LRMS (ESI⁺) m/z calcd forC₁₃₆H₁₉₆Cl₂N₂₂O₄₃S₂ (M+2H⁺)/2=1480.13; found 1480.35. LRMS (ESI⁺) m/zcalcd for C₈₅H₁₁₉ClN₁₄O₃₁S₂(M+2H⁺)/2=965.36; found 965.54.

As a side-product, the mono-substituted Ahx-maytansin derivative of 68was isolated (not depicted). LRMS (ESI⁺) calculated forC₈₅H₁₁₉ClN₁₄O₃₁S₂ ²⁺ m/z 965.36 found 965.54.

Example 56 Synthesis of (39)

To the mono-substituted Ahx-maytansin derivative of 68, isolated as theside-product in example 55, was added a solution of Et₃N (0.7 μL, 0.5mg, 5 μmol) in DMF (1 mL) and a solution of 1.2 mg (1.1 μmol)H-Val-Cit-PABA-MMAF. The mixture was allowed to react overnight and2,2′-(ethylenedioxy)bis(ethylamine) (1 μL, 1.0 mg, 6.7 μmol) was added.After 15 min, the reaction mixture was concentrated. Reversed phase(C18) HPLC chromatography (30-90% MeCN (1% AcOH) in H₂O (1% AcOH)afforded 1.0 mg of the desired product. LRMS (ESI⁺) m/z calcd forC₁₃₇H₂₀₆ClN₂₃O₄₁S₂(M+2H⁺)/2=1464.69; found 1465.66.

Example 57 Determination of HPLC Retention Time

Samples of compounds 19-38 were injected on an HPLC system equipped withan Phenomenex Luna C18(2) 5 t, 150×4.6 mm, 100 Å column and eluted witheither a gradient of 10% MeCN (0.1% TFA)/90% water (0.1% TFA) to 90%MeCN (0.1% TFA)/10% water (0.1% TFA) or a gradient of 10% MeCN/90% 10 mMpotassium phosphate buffer pH 7.4 to 90% MeCN/10% 10 mM potassiumphosphate buffer pH 7.4. The retention times obtained for thesecompounds are depicted in Table 3.

Time Program:

0-12 min: 10% MeCN to 90% MeCN

12-14 min: 90% MeCN

14-15 min: 90% MeCN to 10% MeCN

15-17 min: 10% MeCN

TABLE 3 Retention times of compounds 19-23 and 30-38 on RP-HPLC.retention time (min) compound 0.1% TFA pH 7.4 19 (comp.) 11.4 11.4 2011.6 9.6 21 11.6 9.2 22 (comp.) 12.7 12.6 23 10.6 6.9 30 (comp.) 10.510.6 31 (comp.) 10.1 10.1 32 (comp.)  9.7 9.7 33 10.4 9.3 34 (comp.)11.5 11.5 35 11.6 10.0 36 10.4 9.3 37 11.2 10.5 38 11.1 9.5

Example 58 Competition Conjugation of 17 vs 18 to Trastuzumab(N300C)

Trastuzumab(N300C) (100 μM) was incubated for 2 hours at roomtemperature in PBS with 30 mM EDTA and 1 mM TCEP (10 eq.). Buffer wasexchanged to PBS after which the partially reduced trastuzumab(N300C)(66.7 μM) was incubated with 1.33 mM dehydroascorbic acid (20 eq.). Thereoxidized trastuzumab(N300C) (66.7 μM) was incubated with 0.4 mMmaleimide 17 (6 eq.) and 0.4 mM maleimide 18 (6 eq.). After 1 hourincubation at room temperature the reaction was quenched by adding a5-fold molar excess of N-acetylcysteine. Reaction products were digestedwith Fabricator™ (purchased from Genovis) and analyzed by MS-analysis(AccuTOF). Approximately 45% of the Fc-fragment was conjugated tomaleimide 18 (24297 Da, expected mass=24299) and approximately 35% ofthe Fc-fragment was conjugated to maleimide 17 (24319 Da, expectedmass=24323). The remaining 20% of Fc-fragments consists of variousnonconjugated forms (ranging from 23776 to 23874 Da).

Example 59 Conjugation of 19-39 to 13b or 13c

To a solution of the appropriate trastuzumab(azide)₂ (8.8 μL, 0.2 mg,22.7 mg/ml in Tris buffer pH 7.5 10 mM) was added Tris buffer pH 7.5 10mM (4 μL) and the substrate (2 μL, 2 mM solution in MiliQ+5% DMA). Thereaction was incubated at rt and samples (2 μL) were taken at pre-settime points. These samples were incubated with DTT (2 μL 0.2 M), dilutedwith MiliQ (30 μL) and subjected to MS analysis (AccuTof) to determinethe conjugation efficiency (see Table 1 and 2).

Example 60 Aggregation

The tendency to aggregate was investigated for conjugates of 36, 37 and38 with trastuzumab(F₂-GalNAz)₂ (13c), prepared according to example 59.The ADCs were incubated in PBS pH 7.4 at a concentration of 1 mg/mL at37° C. The level of aggregation was analysed using a Superdex200 PC3.2/30 column (GE Healthcare) after 0, 1 and 2 weeks. The results aredepicted in FIG. 23. Aggregation remained below 1% upon going from thedrug to antibody ratio of 2 (DAR2) of conjugate 36 to the drug toantibody ratio of 4 (DAR4) of conjugate 37, indicating the potential ofsulfamide spacer to compensate the increase of lipophilicity imparted bydoubling the number of payloads. For the bis-sulfamide linker of 38, noaggregation at all was observed. These results are a marked improvementover conventional bioconjugates prepared by alkyne-azide cycloadditionligation.

Example 61 Aggregation

The tendency to aggregate for conjugates of 30 and 36 withtrastuzumab(F₂-GalNAz)₂ (13c), prepared according to example 59, and fortrastuzumab itself, was monitored over a period of 14 days. Theconjugates were stored for 2 weeks at 40° C. at pH 5, and at day 0, 2,7, 10 and 14 the extent of aggregation was determined. The level ofaggregation was analysed using a Superdex200 PC 3.2/30 column (GEHealthcare). The results are depicted in FIG. 24. The conditions withincreased stress (increased T, lower pH) compared to example 61 lead toincreased aggregation. Nevertheless, aggregation was significantlyreduced for the conjugate according to the invention (conjugate of 36with trastuzumab(F₂-GalNAz)₂ (13c)), compared to the comparativebioconjugate of 30 with trastuzumab(F₂-GalNAz)₂ (13c), employing a PEG₄spacer. Notably, trastuzumab itself did not show any tendacy toaggregate, in view of the absence of hydrophobic groups such as a1,2,3-triazole moiety fused to a cyclooctane and the maytansinoidmoiety.

The invention claimed is:
 1. A process for the preparation of abioconjugate, the process comprising the step of reacting a reactivegroup Q¹ of a linker-conjugate with a functional group F¹ of aglycoprotein (B) under condition such that the reactive group Q¹ isreacted with the functional group F¹ of the glycoprotein to covalentlylink the glycoprotein to the linker-conjugate, wherein thelinker-conjugate is a compound comprising a group according to formula(1)

 covalently linked to an alpha-end at one side of the formula (1) and anomega-end at the other side of the formula (1), the alpha-end comprisingthe reactive group Q¹ capable of reacting with the functional group F¹present on the glycoprotein (B) and the omega-end comprising a targetmolecule (D) selected from the group consisting of an active substance,a reporter molecule, a polymer, a solid surface, a hydrogel, ananoparticle, a microparticle and a biomolecule, wherein: the wavy linesindicate covalent linkages to the alpha end and the omega end, a is 0 or1; and R¹ is selected from the group consisting of hydrogen, C₁-C₂₄alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups,C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups, theC₁-C₂₄ alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)arylgroups, C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkylgroups optionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁-C₄ alkyl groups,or R¹ is a second target molecule D, wherein the second target moleculeis optionally connected to N via a spacer moiety; and wherein Q¹ is a(hetero)cyclooctynyl group or a bicycle[6.1.0]non-4-yn-9yl] group, andF¹ is an azide group.
 2. The process according to claim 1, wherein, inthe group according to formula (1), a is
 0. 3. The process according toclaim 1, wherein, in the group according to formula (1), a is
 1. 4. Theprocess according to claim 1, wherein the target molecule is an activesubstance, a reporter molecule or a polymer.
 5. The process according toclaim 1, wherein the reaction is a cycloaddition.
 6. The processaccording to claim 5, wherein the cycloaddition is a Diels-Alderreaction or a 1,3-dipolar cycloaddition.
 7. The process according toclaim 1, wherein the linker-conjugate has formula (4a) or (4b), or asalt thereof:

wherein: a is independently 0 or 1; b is independently 0 or 1; c is 0 or1; d is 0 or 1; e is 0 or 1; f is an integer in the range of 1 to 150; gis 0 or 1; i is 0 or 1; Sp¹ is a spacer moiety; Sp² is a spacer moiety;Sp³ is a spacer moiety; Sp⁴ is a spacer moiety; Z¹ is a connecting groupthat connects Q¹ or Sp³ to Sp², O or C(O) or N(R¹); Z² is a connectinggroup that connects D or Sp⁴ to Sp¹, N(R¹), O or C(O); and R¹ isselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups, C₃-C₂₄alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groups, the C₁-C₂₄alkyl groups, C₃-C₂₄ cycloalkyl groups, C₂-C₂₄ (hetero)aryl groups,C₃-C₂₄ alkyl(hetero)aryl groups and C₃-C₂₄ (hetero)arylalkyl groupsoptionally substituted and optionally interrupted by one or moreheteroatoms selected from O, S and NR³ wherein R³ is independentlyselected from the group consisting of hydrogen and C₁-C₄ alkyl groups;or R¹ is D, -[(Sp¹)_(b)-(Z²)_(e)(Sp⁴)_(i)-D] or-[(Sp²)_(c)-(Z¹)_(d)-(Sp³)_(g)-Q¹], wherein Sp¹, Sp², Sp³, Sp⁴, Z¹, Z²,D, Q¹, b, c, d, e, g and i are as defined above.
 8. The processaccording to claim 7, wherein Sp¹, Sp², Sp³ and Sp⁴ are independentlyselected from the group consisting of linear or branched C₁-C₂₀₀alkylene groups, C₂-C₂₀₀ alkenylene groups, C₂-C₂₀₀ alkynylene groups,C₃-C₂₀₀ cycloalkylene groups, C₅-C₂₀₀ cycloalkenylene groups, C₈-C₂₀₀cycloalkynylene groups, C₇-C₂₀₀ alkylarylene groups, C₇-C₂₀₀arylalkylene groups, C₈-C₂₀₀ arylalkenylene groups and C₉-C₂₀₀arylalkynylene groups, the alkylene groups, alkenylene groups,alkynylene groups, cycloalkylene groups, cycloalkenylene groups,cycloalkynylene groups, alkylarylene groups, arylalkylene groups,arylalkenylene groups and arylalkynylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group of O, S and NR³, wherein R³ is independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl groups,C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynyl groups and C₃-C₂₄ cycloalkylgroups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkylgroups being optionally substituted.
 9. The process according to claim7, wherein Z¹ and Z² are independently selected from the groupconsisting of —O—, —S—, —NR²—, —N═N—, —C(O)—, —C(O)NR²—, —O—C(O)—,—O—C(O)—O—, —O—C(O)—NR², —NR₂—C(O)—, —NR²—C(O)—O—, —NR²—C(O)—NR²—,—S—C(O)—, —S—C(O)—O—, —S—C(O)—NR²—, —S(O)—, —S(O)₂—, —O—S(O)₂—,—O—S(O)₂—O—, —O—S(O)₂—NR²—, —O—S(O)—, —O—S(O)—O—, —O—S(O)—NR²—,—O—NR²—C(O)—, —O—NR²—C(O)—O—, —O—NR²—C(O)—NR²—, —NR²—O—C(O)—,—NR²—O—C(O)—O—, —NR²—O—C(O)—NR²—, —O—NR²—C(S)—, —O—NR²—C(S)—O—,—O—NR²—C(S)—NR²—, —NR²—O—C(S)—, —NR²—O—C(S)—O—, —NR²—O—C(S)—NR²—,—O—C(S)—, —O—C(S)—O—, —O—C(S)—NR²—, —NR²—C(S)—, —NR²—C(S)—O—,—NR²—C(S)—NR²—, —S—S(O)₂—, —S—S(O)₂—O—, —S—S(O)₂—NR²—, —NR²—O—S(O)—,—NR²—O—S(O)—O—, —NR²—O—S(O)—NR²—, —NR²—O—S(O)₂—, —NR²—O—S(O)₂—O—,—NR²—O—S(O)₂—NR²—, —O—NR²—S(O)—, —O—NR²—S(O)—O—, —O—NR²—S(O)—NR²—,—O—NR²—S(O)₂—O—, —O—NR²—S(O)₂—NR²—, —O—NR²—S(O)₂—, —O—P(O)(R²)₂—,—S—P(O)(R²)₂—, —NR²—P(O)(R²)₂— and combinations of two or more thereof,wherein R² is independently selected from the group consisting ofhydrogen, C₁-C₂₄ alkyl groups, C₂-C₂₄ alkenyl groups, C₂-C₂₄ alkynylgroups and C₃-C₂₄ cycloalkyl groups, the alkyl groups, alkenyl groups,alkynyl groups and cycloalkyl groups being optionally substituted. 10.The process according to claim 7, wherein Sp¹, Sp², Sp^(a) and Sp⁴, ifpresent, are independently selected from the group consisting of linearor branched C₁-C₂₀ alkylene groups, the alkylene groups being optionallysubstituted and optionally interrupted by one or more heteroatomsselected from the group consisting of O, S and NR³, wherein R³ isindependently selected from the group consisting of hydrogen and C₁-C₄alkyl groups, and wherein Q¹ is a group of formula (9q), (9n), (9o), or(9p):

wherein U is O or NR⁹, and wherein the aromatic rings in (9n) areoptionally O-sulfonylated at one or more positions and the rings of (9o)and (9p) are optionally halogenated at one or more positions.
 11. Theprocess according to claim 1, wherein the glycoprotein is an antibody.12. The process according to claim 11, wherein the antibody specificallybinds an antigen.
 13. The process according to claim 12, wherein theantigen is a cancer related antigen.
 14. The process according to claim1, wherein the bioconjugate is an antibody-drug-conjugate.
 15. Abioconjugate obtained by the process according to claim 1.